Genes Cells, 1997 Dec, 2(12), 725 - 34 Regions of the Escherichia coli primary sigma factor sigma70 that are involved in interaction with RNA polymerase core enzyme; Nagai H et al.; BACKGROUND: The sigma factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation . Most sigma factors share several regions with significant homology in their amino acid sequences (regions 1-4) . Some primary sigma factors carry a large nonconserved segment between regions 1 and 2 . The binding of an sigma factor to the core enzyme alters the structure and properties of the sigma factor, but little is known about the binding mechanism and subsequent reactions . In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli sigma70 by binding to core enzyme and promoter DNA . RESULTS: A segment between regions 1.1 and 1.2, and that in region 3.2, were preferentially cleaved by hydroxyl radicals . Upon binding to the core enzyme, segments in regions 1.1, 2, 3 and 4 were substantially protected, while cleavage at a small segment in region 4.2 was weakly enhanced . In a binary complex of holoenzyme and promoter DNA, additional segments in regions 2.4 and 4.2 were protected, while the protection at region 1.1 disappeared . The nonconserved acidic region of sigma70 in the holoenzyme became hypersensitive upon binding to DNA . CONCLUSIONS: These results suggest that not only the conserved region 2, but also regions 1.1, 3 and 4 of the sigma factor are involved in binding to the core enzyme . The nonconserved acidic region is likely to be more exposed by further binding of sigma factor to promoter DNA. Genetics, 1998 Mar, 148(3), 963 - 73 The yeast HSM3 gene acts in one of the mismatch repair pathways; Fedorova IV et al.; Mutants with enhanced spontaneous mutability (hsm) to canavanine resistance were induced by N-methyl-N-nitrosourea in Saccharomyces cerevisiae . One bearing the hsm3-1 mutation was used for this study . This mutation does not increase sensitivity to the lethal action of different mutagens . The hsm3-1 mutation produces a mutator phenotype, enhancing the rates of spontaneous mutation to canavanine resistance and reversions of lys1-1 and his1-7 . This mutation increases the rate of intragenic mitotic recombination at the ADE2 gene . The ability of the hsm3 mutant to correct DNA heteroduplex is reduced in comparison with the wild-type strain . All these phenotypes are similar to ones caused by pms1, mlhl and msh2 mutations . In contrast to these mutations, hsm3-1 increases the frequency of ade mutations induced by 6-HAP and UV light . Epistasis analysis of double mutants shows that the PMS1 and HSM3 genes control different mismatch repair systems . The HSM3 gene maps to the right arm of chromosome II, 25 cM distal to the HIS7 gene . Strains that bear a deleted open reading frame YBR272c have the genetic properties of the hsm3 mutant . The HSM3 product shows weak similarity to predicted products of the yeast MSH genes (homologs of the Escherichia coli mutS gene) . The HSM3 gene may be a member of the yeast MutS homolog family, but its function in DNA metabolism differs from the functions of other yeast MutS homologs. Int J Cancer, 1998 Apr 13, 76(2), 232 - 9 Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas; Helfrich W et al.; We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3epsilon chain of human TCR/CD3 complex . The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains . Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escherichia coli by IMAC chromatography . The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells . The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma-derived BsF(ab')2 Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr 51Cr-release assay . This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell-based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo. Protein Sci, 1998 Mar, 7(3), 789 - 93 Tautomeric state and pKa of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system; Garrett DS et al.; The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range 1H-15N correlation spectroscopy . The active site His 189 is phosphorylated at the Nepsilon2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189 . Because the neutral form of unphosphorylated His 189 is in the Ndelta1-H tautomer, and its Nepsilon2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g+ conformer in the unphosphorylated state to the g- conformer in the phosphorylated state. Protein Sci, 1998 Mar, 7(3), 746 - 57 Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein; Crabb JW et al.; Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Muller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration . The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa . CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein . Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively . The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture . Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass . Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP . Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties . Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal . These recombinant preparations provide valid models for human CRALBP structure-function studies. Protein Sci, 1998 Mar, 7(3), 637 - 48 Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides; Nihalani D et al.; The mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1-59 or SK1-63) enhance the low plasminogen (PG)-activating ability of other fragments, namely SK64-386, SK60-414, SK60-387, and SK60-333 (reported previously), has been investigated using a synthetic peptide approach . The addition of either natural SK1-59, or chemically synthesized SK16-59, at saturation (about 500-fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60-414 or SK56-414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2- and 2.5-fold, respectively, of that generated by equimolar mixtures of native SK and HPG . Although in the absence of SK1-59 equimolar mixtures of SK56-414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N-terminal region lay mainly in imparting in SK56-414 an enhanced ability for PG activation . The ability of various synthetic peptides derived from the amino-terminal region (SK16-51, SK16-45, SK37-59, SK1-36, SK16-36, and SK37-51) to (1) complement equimolar mixtures of SK56-414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56-414 and HPG by SK16-59, and (3) directly inhibit PG activation by the 1:1 SK-HPG activator complex was tested . Apart from SK16-59, SK16-51, and 16-45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56-414 survived even in the smaller SK-peptides, viz., SK37-59 and SK37-51 . However, this ability was abolished upon specifically mutating the sequence -LTSRP-, present at position 42-46 in native SK . Although SK16-51 retained virtually complete ability for potentiation of PG activation in comparison to SK16-59 or SK1-59, this ability was reduced by approximately fourfold in the case of SK16-45, and completely abolished upon further truncation of the C-terminal residues to SK16-36 or SK1-36 . Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG . This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25-59) in which the sequence corresponding to this putative locus (residues 16-36) was truncated at the middle . On the other hand, fragments SK37-51 and SK37-59 did not show any inhibition of the PG activation by native activator complex . Taken together, these findings strongly support a model of SK action wherein the HPG binding site resident in the region 37-51 helps in anchoring the N-terminal domain to the strong intermolecular complex formed between HPG and the region 60-414 . In contrast, the site located between residues 16 and 36 is qualitatively more similar to the previously reported PG interacting site (SK254-273) present in the core region of SK, in being involved in the relatively low-affinity enzyme-substrate interactions of the activator complex with PG during the catalytic cycle. Protein Sci, 1998 Mar, 7(3), 605 - 10 Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases; Korolev S et al.; Three helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep . PcrA and Rep belong to the same super-family of helicases (SF1) and are structurally very similar . In contrast, the HCV helicase belongs to a different super-family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases . Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super-families . The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases. Eur J Oral Sci, 1998 Jan, 106 Suppl 1, 324 - 30 Sequential expression of an amelin gene in mesenchymal and epithelial cells during odontogenesis in rats; Fong CD et al.; Novel mRNA isoforms encoding the enamel matrix proteins amelin-1, amelin-2 and ameloblastin have been recently described . We have applied detailed immunohistochemical as well as non-radioactive in situ hybridization analyses to follow amelin-1 expression in developing rat incisors and molars . We constructed an expression vector, overproduced recombinant amelin in Escherichia coli and prepared an antibody . In addition to the previously reported amelin mRNA expression patterns in ameloblasts, the amelin message was also detected in pulpal mesenchymal cells including preodontoblasts and young odontoblasts . The signal in these cells persisted until deposition of mantle dentin became evident . The immunolocalization of amelin-1 in preodontoblasts and ameloblasts essentially followed the pattern of mRNA expression . The most intense staining was found in the enamel matrix adjacent to secretory ameloblasts . Focal accumulations of immunoreactive material were found at the dentinoenamel junction during the maturation stage . Also, using 5'-RACE (Rapid Amplification of cDNA Ends) we could confirm only amelin-1 and ameloblastin messages in the total RNA pool from rat molars and conclude that amelin-2 is a truncated form of ameloblastin . The sequential expression of amelin in mesenchymal and epithelial cells suggests it plays a role in cell differentiation during early tooth development. FEBS Lett, 1998 Mar 20, 425(1), 161 - 5 Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the pMV158-encoded plasmid transcriptional repressor protein CopG; Gomis-Ruth FX et al.; Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control . A new procedure for overproduction and purification of the protein has been developed . The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter . Purified CopG protein has been crystallized using the sitting-drop vapor diffusion method . The crystals, belonging to orthorhombic space group C222(1) (cell constants a = 67.2 A, b = 102.5 A, c = 40.2 A), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7 . Complete diffraction data up to 1.6 A resolution have been collected . Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 A3/Da). FEBS Lett, 1998 Mar 20, 425(1), 134 - 6 Lipopolysaccharide-caused fragmentation of individual microtubules in vitro observed by video-enhanced differential interference contrast microscopy; Bohm KJ et al.; Microtubule disassembly is commonly believed to be a process of endwise tubulin dimer release . The present study demonstrates by video interference contrast microscopy that Escherichia coli lipopolysaccharide (LPS) caused microtubule disassembly in vitro by both endwise shortening and fragmentation . In contrast, the microtubules were only shortened from their ends in the presence of DNA, used as another example of a macromolecular microtubule effector . LPS-caused microtubule fragmentation was confirmed by transmission electron microscopy . Because of its ability to induce both fragmentation and endwise shortening, LPS, which is involved in sepsis pathogenesis, has to be regarded as a highly active microtubule-destabilizing agent. FEBS Lett, 1998 Mar 20, 425(1), 101 - 4 Can grafting of an octapeptide improve the structure of a de novo protein? Aphasizheva IY, Dolgikh DA, Abdullaev ZK, Uversky VN, Kirpichnikov MP, Ptitsyn OB. Structural properties and conformational stability of de novo proteins -- albebetin and albeferon (albebetin with a grafted interferon fragment) -- were studied by means of CD spectroscopy, gel filtration and urea-induced unfolding . The results allow us to conclude that albebetin possesses the properties of the molten globule state . Grafting of the octapeptide to the N-terminus of this de novo protein affects its structure . We show here that albeferon maintains a secondary structure content of albebetin; it becomes more compact and much more stable toward urea-induced unfolding as compared to albebetin and even possesses some weak tertiary structure (at least around Tyr7) . This means that the structure of the artificial protein albebetin can be improved by a simple procedure of octapeptide grafting to its N-terminus. Biochim Biophys Acta, 1998 Feb 17, 1382(2), 333 - 8 Studies on the urea cycle enzyme ornithine transcarbamylase using heavy atom isotope effects; Parmentier LE et al.; Ornithine transcarbamylase (OTCase) catalyzes the reaction between L-ornithine and carbamyl phosphate in the first step of the urea cycle . 13C isotope effects were measured in carbamyl phosphate, using OTCase obtained from E . coli in a one-column purification which yielded 30 mg of very pure enzyme from 51 of cell culture . At near zero L-ornithine, the 13C kinetic isotope effect was 1.0095, at high levels of L-ornithine (86 mM) the 13C kinetic isotope effect was unity, and 0.83 mM ornithine was found to eliminate half the isotope effect . These results are indicative of an ordered kinetic mechanism in which carbamyl phosphate binds to the enzyme before L-ornithine . Similar experiments were performed using the slow substrate L-lysine in place of L-ornithine . At 90 mM L-lysine the 13C kinetic isotope effect was large, 1.076 . This value is most likely the intrinsic kinetic isotope effect with this substrate, and the chemistry of the enzyme catalyzed reaction has become rate limiting. Biochim Biophys Acta, 1998 Feb 17, 1382(2), 230 - 42 Identification and characterization of receptors for riboflavin carrier protein in the chicken oocyte . Role of the phosphopeptide in mediating receptor interaction; Sooryanarayana et al.; Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg and the serum of laying birds and other animals . We have investigated the binding of chicken RCP (cRCP) to membranes prepared from the whole chicken oocytes . RCP binding had an absolute requirement for calcium, with an affinity (Kd 10(-8) M) high enough to be physiologically relevant . Ligand blotting experiments using labeled RCP and vitellogenin, with proteins solubilized from oocyte membranes, indicated that RCP and vitellogenin bound specifically to three proteins of Mr 380, 260 and 110 kDa . Vitellogenin also bound to proteins of Mr 515 kDa and 97 kDa, similar in size to those identified by receptor associated protein of RAP . Reduced and carboxyamidated RCP inhibited the binding of 125I-labeled RCP to chicken oocyte membranes, but recombinant RCP expressed in E . coli, and dephosphorylated RCP, failed to interact with the receptors, indicating that post-translational modifications were necessary for ligand-receptor interaction . The purified phosphopeptide, prepared from tryptic digests of egg white RCP, was able to inhibit the binding of RCP to the receptor proteins, with an affinity comparable to native RCP indicating that the phosphopeptide sequence present in RCP serves as the focal point for RCP-receptor interactions. J Photochem Photobiol B, 1998 Feb, 42(2), 109 - 24 Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions; Favre A et al.; In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies . These thio residues such as 4-thiouracil found in E . coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated . Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties . Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines . From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized . Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct . A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine . The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines . The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit . Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome . Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals . Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them . Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes). Biophys Chem, 1998 Feb 16, 70(2), 101 - 8 A thermodynamic study of the binding of linear and cyclic oligosaccharides to the maltodextrin-binding protein of Escherichia coli; Thomson J et al.; Isothermal titration calorimetric (ITC) studies over a range of temperatures of the binding of maltose, maltotriose, maltotetraose and beta-cyclodextrin to the maltodextrin-binding protein (MBP) of Escherichia coli are reported . The binding constants of maltose, maltotriose and beta-cyclodextrin are not very different, namely 8.7 x 10(5), 13.0 x 10(5) and 2.55 x 10(5) M-1, respectively at 25 degrees C . The calorimetric data obtained with maltotetraose cannot be interpreted in terms of a definite binding constant . The binding of maltose and maltotriose is endothermic with a large entropy increase while that of beta-cyclodextrin is exothermic, with a smaller entropy increase . The binding of maltotetraose was endothermic or exothermic depending on the temperature. Mutat Res, 1998 Feb, 407(1), 47 - 53 Modification of enzyme sulfhydryl groups suppresses UV-induced mutagenesis depending on the nucleotide excision repair system in Escherichia coli B/r WP2; Nakamura Y et al.; S-Methyl methanethiosulfonate (MMTS), which was isolated from cauliflower (Brassica oleracea var . botrytis) homogenate as a potent bio-antimutagen, has been used as an enzyme-sulfhydryl (SH) temporary blocking agent in modification studies of enzyme activities . We examined whether 23 kinds of MMTS-related compounds have a suppressing effect on UV mutagenesis in Escherichia coli B/r WP2 . Disulfide derivatives of diphenyl, 2.2'-dipyridine and 4.4'-dipyridine, and N-ethyl maleimide (NEM), which temporarily or tightly block sulfhydryl (SH)-groups, showed similar suppressing effect in E . coli B/r WP2, but not in WP2s hcr- (uvrA-) in the range of nanomolar/plate as MMTS previously did . Cystamine sulfate, methyl methylsulfinylmethyl sulfide and S-methyl-L-cysteinesulfoxide moderately suppressed, and diallyl disulfide and glutathione (oxidized form) weakly suppressed UV mutagenesis in E . coli B/r WP2 in the range of micromolar/plate . MMTS and phorone, a glutathione (GSH)-depleting agent, lowered the intracellular GSH level in E . coli B/r WP2, but phorone did not inhibit UV-induced mutation . These results indicate that the target for SH-modification is enzyme-SHs but not GSH, and that the direct or indirect modification of enzyme-activity by SH-blocking might be involved in the antimutagenesis through a pathway associated with the DNA-excision repair system. Mutat Res, 1998 Feb, 407(1), 11 - 24 Extrachromosomal unequal homologous recombination and gene conversion in simian kidney cells: effects of UV damage; Gening L et al.; Shuttle plasmid vectors containing the SV40 origin of replication and tandem neo genes with distally placed non-overlapping deletions were used to study the effects of DNA damage on extrachromosomal homologous recombination in simian kidney cells . DNA was introduced into COS7 cells by a lipofectin-mediated transfection procedure and recombination was assessed by analyzing the structure of plasmids . Recombinational events observed included unequal homologous recombination (triplication), gene conversion, double reciprocal recombination, deletion (pop-outs), gene amplification (4-6 copies), and multimerization . Triplication, an event that previously had not been reported in association with extrachromosomal recombination, predominated in experiments with undamaged vectors . The recombination frequency (NeoR/AmpR) of vectors randomly damaged by UV irradiation was essentially unchanged; however, the relative number of triplication events decreased significantly . Selective damage in one of the two neo genes increased the relative frequency of gene conversion . The experimental system developed for use in this study detects all major homologous recombination events observed in chromosomal direct repeat sequences in mammalian cells and yeast and should prove valuable for future studies of homologous recombination in mammalian cells. Genetics, 1998 Mar, 148(3), 1091 - 108 The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near-standard genetic code; Beagley CT et al.; The circular, 17,443 nucleotide-pair mitochondrial (mt) DNA molecule of the sea anemone, Metridium senile (class Anthozoa, phylum Cnidaria) is presented . This molecule contains genes for 13 energy pathway proteins and two ribosomal (r) RNAs but, relative to other metazoan mtDNAs, has two unique features: only two transfer RNAs (tRNA(f-Met) and tRNA(Trp)) are encoded, and the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes each include a group I intron . The COI intron encodes a putative homing endonuclease, and the ND5 intron contains the molecule's ND1 and ND3 genes . Most of the unusual characteristics of other metazoan mtDNAs are not found in M . senile mtDNA: unorthodox translation initiation codons and partial translation termination codons are absent, the use of TGA to specify tryptophan is the only genetic code modification, and both encoded tRNAs have primary and secondary structures closely resembling those of standard tRNAs . Also, with regard to size and secondary structure potential, the mt-s-rRNA and mt-1-rRNA have the least deviation from Escherichia coli 16S and 23S rRNAs of all known metazoan mt-rRNAs . These observations indicate that most of the genetic variations previously reported in metazoan mtDNAs developed after Cnidaria diverged from the common ancestral line of all other Metazoa. Genetics, 1998 Mar, 148(3), 1043 - 54 Organization of chromosome ends in Ustilago maydis . RecQ-like helicase motifs at telomeric regions; Sanchez-Alonso P et al.; In this study we have established the structure of chromosome ends in the basidiomycete fungus Ustilago maydis . We isolated and characterized several clones containing telomeric regions and found that as in other organisms, they consist of middle repeated DNA sequences . Two principal types of sequence were found: UTASa was highly conserved in nucleotide sequence and located almost exclusively at the chromosome ends, and UTASb was less conserved in nucleotide sequence than UTASa and found not just at the ends but highly interspersed throughout the genome . Sequence analysis revealed that UTASa encodes an open reading frame containing helicase motifs with the strongest homology to RecQ helicases; these are DNA helicases whose function involves the maintenance of genome stability in Saccharomyces cerevisiae and in humans, and the suppression of illegitimate recombination in Escherichia coli . Both UTASa and UTASb contain a common region of about 300 bp located immediately adjacent to the telomere repeats that are also found interspersed in the genome . The analysis of the chromosome ends of U . maydis provides information on the general structure of chromosome ends in eukaryotes, and the putative RecQ helicase at UTASa may reveal a novel mechanism for the maintenance of chromosome stability. FEBS Lett, 1998 Mar 13, 424(3), 253 - 6 Iron attenuates nitric oxide level and iNOS expression in endotoxin-treated mice; Komarov AM et al.; The effect of exogenous Fe-citrate complex (Fe doses of 120 and 240 micromol/kg) on nitric oxide (NO) production in vivo has been studied in blood and liver tissue of endotoxin-treated mice . Fe-citrate complex was administered to mice subcutaneously at the same time with intravenous injection of Escherichia coli lipopolysaccharide (LPS) . Iron-dependent decrease in NO2-/NO3- and nitrosyl hemoglobin levels in blood of animals was detected at 6 h after LPS administration, suggesting systemic attenuation of NO generation . NO production in the liver tissue of LPS-treated mice was decreased after Fe administration judging from the amount of mononitrosyl-iron complexes formed in the tissue by diethyldithiocarbamate . The iNOS protein determination in the liver tissue of LPS-treated mice demonstrated iron-dependent inhibition of iNOS expression . We have found previously that exogenous iron does not affect systemic NO level when it is given at 6 h after LPS injection, i.e . after iNOS expression . This is a first report demonstrating iron-dependent iNOS down-regulation in endotoxin-treated mice. FEBS Lett, 1998 Mar 13, 424(3), 121 - 6 Analysis of three human interleukin 5 structures suggests a possible receptor binding mechanism; Verschelde JL et al.; We compared three crystal structures of human interleukin 5 (hIL5) expressed in either E . coli (hIL5E.coli), Sf9 cells (hIL5sf9) or Drosophila cells (hIL5Drosophila) . The dimeric hIL5 structures show subtle but significant conformational differences which are probably a consequence of the different crystallization conditions trapping this protein into one of two states . We refer to these two distinct conformations as the 'open' and 'tight' state, according to the packing around the cleft between the two subunits . We hypothesize that these two stable conformational states reflect the structure of the free or receptor bound hIL5. Trends Biochem Sci, 1998 Feb, 23(2), 68 - 73 Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms; Netzer WJ et al.; Recent findings suggest that a combination of chaperonin-assisted and unassisted mechanisms operate in protein folding in the cytosol . While nascent chain-binding chaperones, such as Hsp70, could have a general role in maintaining the folding competence of translating polypeptide chains, the contribution of the cylindrical chaperonin complexes to overall folding is limited to a subset of aggregation-sensitive polypeptides . The majority of bacterial proteins are relatively small and they are synthesized rapidly and folded independently of the chaperonin GroEL in a posttranslational manner . Eukaryotes have a proportionally larger number of multi-domain proteins than bacteria . The individual domains of these proteins can be folded cotranslationally and sequentially . The use of this mechanism explains how large proteins fold independently of a chaperonin and could have been crucial in the evolution of a wide array of modular polypeptides in eukaryotes. J Bacteriol, 1998 Apr, 180(7), 1970 - 2 Growth rate-dependent accumulation of RNA from plasmid-borne rRNA operons in Escherichia coli; Stevenson BS et al.; Inadequate regulation of the expression of additional plasmid-borne rRNA operons in Escherichia coli was exaggerated at slow growth rates, resulting in increases of approximately 100% for RNA concentration and 33% for doubling time . These observations are consistent with the hypothesis that multiple rRNA operons constitute a metabolic burden at slow growth rates. J Bacteriol, 1998 Apr, 180(7), 1947 - 50 The active-site cysteines of the periplasmic thioredoxin-like protein CcmG of Escherichia coli are important but not essential for cytochrome c maturation in vivo; Fabianek RA et al.; A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli . The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain . A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes . Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c550 expressed in E . coli . This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG. J Bacteriol, 1998 Apr, 180(7), 1944 - 6 Levels of the Vsr endonuclease do not regulate stationary-phase reversion of a Lac- frameshift allele in Escherichia coli; Foster PL et al.; Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells . Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac- frameshift allele, but the absence of Vsr has no effect . Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual. J Bacteriol, 1998 Apr, 180(7), 1929 - 38 Escherichia coli mrsC is an allele of hflB, encoding a membrane-associated ATPase and protease that is required for mRNA decay; Wang RF et al.; The mrsC gene of Escherichia coli is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive mrsC505 allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L . L . Granger et al., J . Bacteriol . 180:1920-1928, 1998) . The cloned mrsC gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain . mrsC is identical to the independently identified ftsH gene except for three additional amino acids at the N terminus (T . Tomoyasu et al., J . Bacteriol . 175:1344-1351, 1993) . The purified protein had a Km of 28 microM for ATP and a Vmax of 21.2 nmol/microg/min . An amino-terminal glutathione S-transferase-MrsC fusion protein retained ATPase activity but was not biologically active . A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (mrsC201) abolished the complementation of the mrsC505 mutation, confirming that the ATPase activity is required for MrsC function in vivo . In addition, the mrsC505 allele conferred a temperature-sensitive HflB phenotype, while the hflB29 mutation promoted mRNA stability at both 30 and 44 degrees C, suggesting that the inviability associated with the mrsC505 allele is not related to the defect in mRNA decay . The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in E . coli. J Bacteriol, 1998 Apr, 180(7), 1920 - 8 The Escherichia coli mrsC gene is required for cell growth and mRNA decay; Granger LL et al.; We have identified a gene in Escherichia coli that is required for both the normal decay of mRNA and RNA synthesis . Originally designated mrsC (mRNA stability), the mrsC505 mutation described here is, in fact, an allele of the hflB/ftsH locus (R.-F . Wang et al., J . Bacteriol . 180:1929-1938, 1998) . Strains carrying the thermosensitive mrsC505 allele stopped growing soon after the temperature was shifted to 44 degrees C but remained viable for several hours . Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min . At 44 degrees C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the mrsC505 single mutant . In an rne-1 mrsC505 double mutant, the average half-life was 19.8 min . Inactivating mrsC significantly increased the half-lives of the trxA, cat, secG, and kan mRNAs, particularly in an mrsC505 pnp-7 rnb-500 rne-1 multiple mutant . In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of mrsC505 multiple mutants at 44 degrees C . These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex. J Bacteriol, 1998 Apr, 180(7), 1841 - 7 Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli; Ault-Riche D et al.; A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP . We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light . This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein . A simplified procedure for preparing polyP synthesized by polyP kinase is also described . Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP . By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels . Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB. J Bacteriol, 1998 Apr, 180(7), 1814 - 21 Identification and function of the pdxY gene, which encodes a novel pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate biosynthesis in Escherichia coli K-12; Yang Y et al.; pdrK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5'-phosphate (PLP) coenzyme biosynthesis . The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase . Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase . PdxY was first identified by its homology to PdxK in searches of the complete E . coli genome . Minimal clones of pdxY+ overexpressed PL kinase specific activity about 10-fold . We inserted an omega cassette into pdxY and crossed the resulting pdxY::omegaKan(r) mutation into the bacterial chromosome of a pdrB mutant, in which de novo PLP biosynthesis is blocked . We then determined the growth characteristics and PL and PN kinase specific activities in extracts of pdxK and pdxY single and double mutants . Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities . Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6 vitamer kinases in E . coli and that their function is confined to the PLP salvage pathway . Last, we show that pdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNA(Tyr) synthetase) in a multifunctional operon . pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in the tyrS-pdxY intercistronic region. J Bacteriol, 1998 Apr, 180(7), 1808 - 13 The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA; Qian Q et al.; tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms . In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37) . We have isolated a mutant of E . coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species . These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37 . We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E . coli chromosomal map . The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain . The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species . We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC. J Bacteriol, 1998 Apr, 180(7), 1803 - 7 Promoter characterization and constitutive expression of the Escherichia coli gcvR gene; Ghrist AC et al.; The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv) . In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein . Primer extension analysis of the gcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon . The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis . Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively . In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR . From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon . However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the bcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence . These results show that gcvR and bcp do not form an operon . Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression. J Bacteriol, 1998 Apr, 180(7), 1786 - 92 Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum; Patel JB et al.; Histoplasma capsulatum is a pathogenic fungus that exists in two distinct forms . The saprophytic mycelial phase inhabits moist soil environments; once inhaled, hyphae and conidia convert to a unicellular yeast phase that is capable of parasitizing macrophage phagolysosomes . Yeasts cultures, but not mycelial cultures, release large quantities of a calcium-binding protein (CBP) which may be important in calcium acquisition during intracellular parasitism . In this study, we show that the gene encoding CBP (CBP1) is transcriptionally regulated . To identify promoter sequences that are important for yeast phase-specific activity, we created a series of fusions between successively truncated CBP1 5' untranslated regulatory sequences and the Escherichia coli lacZ gene . The fusions were constructed on a telomeric shuttle plasmid that can replicate autonomously in the fungus . By assaying for beta-galactosidase activity from H . capsulatum transformants, we identified a 102-bp region that mediates promoter activation and yeast phase promoter activity . Base pair substitution analysis suggests that the sequences between 839 and 877 bp upstream of the start codon are the most important for this positive regulation. J Bacteriol, 1998 Apr, 180(7), 1777 - 85 Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP (cAMP)-cAMP receptor protein complex; Peekhaus N et al.; The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression . Thus, gluconate is both an inducer and a repressor of gntT expression since gluconate is a catabolite-repressing sugar . In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT . Indeed, GntR binds to two consensus gnt operator sites; one overlaps the -10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site . The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites . Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration . Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntT expression which is independent of catabolite repression and binding of GntR to the operator sites . This novel response of gntR mutants to the inducer is termed ultrarepression . Transcription of gntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at -71 upstream of the gntT transcription start site. J Bacteriol, 1998 Apr, 180(7), 1766 - 70 Involvement of recF, recO, and recR genes in UV-radiation mutagenesis of Escherichia coli; Liu YH et al.; The recF, recO, and recR genes were originally identified as those affecting the RecF pathway of recombination in Escherichia coli cells . Several lines of evidence suggest that the recF, recO, and recR genes function at the same step of recombination and postreplication repair . In this work, we report that null mutations in recF, recO, or recR greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp- (trpE65) to a Trp+ phenotypes . Introduction of the defective lexA51 mutation {lexA51(Def)} and/or UmuD' into recF, recO, and recR mutants failed to restore normal UVM in the mutants . On the other hand, the presence of recA2020, a suppressor mutation for recF, recO, and recR mutations, restored normal UVM in recF, recO, and recR mutants . These results indicate an involvement of the recF, recO, and recR genes and their products in UVM, possibly by affecting the third role of RecA in UVM. J Bacteriol, 1998 Apr, 180(7), 1662 - 72 Characterization of the cvaA and cvi promoters of the colicin V export system: iron-dependent transcription of cvaA is modulated by downstream sequences; Boyer AE et al.; Secretion of the Escherichia coli toxin colicin V was previously determined to be iron regulated via the Fur (ferric uptake regulator) protein, based on studies in fur mutants . The iron dependence of transcription and expression of cvaA, which encodes a transporter accessory protein, and cvi, encoding the colicin V immunity protein, was assessed under conditions of iron excess or depletion . Immunoblots showed that production of both Cvi and CvaA is iron dependent . The iron-dependent transcriptional start for cvaA identified by primer extension and S1 nuclease analysis, P1, lies 320 bp upstream of the translational start and is associated with a newly identified Fur binding site . Beta-galactosidase activity in transcriptional lacZ fusions with the P1 promoter alone is higher than with downstream sequences present and is induced 10-fold by iron depletion . Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by iron depletion fivefold . Including subsequent downstream sequences, however, down-modulates overall transcription from P1 almost fourfold . Deletion of a long stem-loop structure in this region alleviates the down-modulation by increasing transcription, indicating that the sequences or structure of this element may contribute to this down-regulation . Characterization of the cvi promoter by primer extension showed that it resides where predicted, about 50 bp upstream of cvi associated with a previously identified Fur binding site . The cvi promoter is also inducible by iron depletion . The modulating sequences from cvaA were placed downstream of the cvi promoter to test their effects in transcriptional fusions of the cvi promoter to lacZ . The fusion results showed that these sequences also modulate transcription of the cvi promoter in a manner similar to that of the cvaA promoter . The potential for up- and down-regulation within the long untranslated region downstream of the cvaA promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes. Cancer Res, 1998 Apr 1, 58(7), 1358 - 62 Adenoviral-mediated transfer of a heat-inducible double suicide gene into prostate carcinoma cells; Blackburn RV et al.; Tumor cells that express a fusion gene comprised of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (TK) sequences exhibit activation of and subsequent killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir (Rogulski et al., Hum . Gene Ther., 8: 73-85, 1997) . To target localized expression of this therapeutic gene, we have constructed a recombinant adenovirus containing the CD-TK fusion gene under the control of a human inducible heat shock protein 70 promotional sequence . Strong expression of the fusion gene product was induced by heating at 41 degrees C for 1 h . Expression levels obtained were dependent on the multiplicity of infection used and the incubation time after heat shock . Heat-induced expression of the CD-TK protein significantly reduced the survival of PC-3 cells in the presence of both 5-fluorocytosine and ganciclovir . These studies represent a novel form of gene therapy for the transduction and regulation of a double suicide gene in tumor cells and may provide a unique application for hyperthermia in cancer therapy. Bull Cancer, 1997 Nov, 84(11), 1047 - 52 {Gene therapy of a model of glioblastoma in rats using adenovirus vector encoding the HSVtk gene}; Quillien V et al.; Our aim was to test the therapeutic effects of adenovirus-mediated gene therapy in an animal model of brain tumor which was obtained by injection of 9L gliosarcoma cells into the caudate nucleus of rat brains . Seven days after the implantation of tumor cells, adenovirus vectors bearing the Escherichia coli beta galactosidase gene (ADV beta-gal) or the herpes simplex virus thymidine kinase gene (ADVtk) were stereotactically injected in the tumor . Injection of the ADV beta gal resulted in the expression of the marker gene in 61% of the animals . Transfer of the ADVtk was followed, 3 days later, by intraperitoneal injection of ganciclovir (GCV) for 10 days . A control group was treated with saline instead of GCV . We observed a significant regression of the tumors in 50% of the rats treated with ADVtk and GCV as compared with control animals . In 4 cases out of 6, the tumor completely disappeared after treatment . These results demonstrate the potential efficacy of adenovirus-mediated transfer of the HSVtk gene following by GCV administration for the treatment of glioblastomas. J Surg Res, 1998 Jan, 74(1), 34 - 8 Endotoxin temporarily impairs canine colonic absorption of water and sodium; Cullen JJ et al.; Diarrhea is a common manifestation of sepsis . We hypothesized that endotoxin may impair colonic absorption of water and electrolytes, an effect which may be related to altered liquid transit in the colon . Five dogs underwent construction of 50-cm colonic Thiry-Vella fistulas (TVF) . Following recovery, absorption studies were performed by perfusing the TVF with an isotonic solution at 2.9 ml/min containing polyethylene glycol (5 g/L) . Fasting and postprandial colonic absorption of water, electrolytes, and glucose were determined . Liquid transit was assessed by bolus of a nonabsorbable marker (PSP) instilled into the proximal end of the TVF . Following completion of the baseline studies, each dog was given a single dose of Escherichia coli lipopolysaccharide 200 micrograms/kg i.v . and the studies were repeated daily for the next 3 days . Following endotoxin bolus, colonic absorption of water and sodium were decreased during fasting, while postprandial colonic absorption of water was also decreased . Colonic absorption of water and sodium returned to baseline values on postendotoxin day 2 . Colonic secretion of potassium was decreased on postendotoxin days 1 and 3 in both the fasting and the fed periods . Fasting and postprandial liquid transit was also rapid on postendotoxin day 1, which correlated with the decreased absorption seen on that day . Liquid transit returned to baseline values on postendotoxin day 2 . We conclude that endotoxin temporarily impairs postprandial colonic absorption, which may be due to the rapid liquid transit that occurs . These effects may contribute to the diarrhea seen during and after septic episodes. Gut, 1998 Feb, 42(2), 200 - 7 Rapid modulation of electrolyte transport in Caco-2 cell monolayers by enteropathogenic Escherichia coli (EPEC) infection; Collington GK et al.; BACKGROUND AND AIMS: The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhoea remains uncertain . EPEC adhere to enterocytes and transduce signals which produce a characteristic "attaching and effacing" (A/E) lesion in the brush border membrane . The present in vitro study was designed to determine whether signal transduction by EPEC also influences electrolyte transport . METHODS: Caco-2 cell monolayers were rapidly infected with wild type EPEC strain E2348/69, or the signal transduction-defective mutant 14.2.1(1), and mounted in Ussing chambers . RESULTS: Strain E2348/69 stimulated a rapid but transient increase in short circuit current (Isc) which coincided with A/E lesion formation; this Isc response was absent on infection with strain 14.2.1(1) . While the initial rise in Isc induced by E2348/69 was partially (approximately 35%) dependent on chloride, the remainder possibly represents an influx of sodium and amino acid(s) across the apical membrane . CONCLUSIONS: The study directly shows that, after initial adhesion, EPEC induce major alterations in host cell electrolyte transport . The observed Isc responses indicate a rapid modulation of electrolyte transport in Caco-2 cells by EPEC, including stimulation of chloride secretion, for which signal transduction to host cells is a prerequisite. Int J Clin Pract . 1997 Nov-Dec;51(8):531. Scrotal suppuration following appendicitis; Pal KM; Inguinoscrotal sepsis following intraperitoneal infection is a recognised but infrequent complication . Incidence of this condition seems to have decreased because of the routine use of broad spectrum antibiotics . It is believed that a persistent communication between the peritoneal cavity and scrotum, in the form of a patent tunic vaginalis, is needed for this complication . There may or may not be an associated hernia . Intrascrotal sepsis can lead to testicular loss from vascular thrombosis . The treatment of choice is early operative drainage. Int J Clin Pract, 1997 Oct, 51(7), 468 - 70 Emphysematous pyelonephritis responding to medical therapy; Punnose J et al.; We describe two type 2 diabetic patients with unilateral emphysematous pyelonephritis who responded to medical treatment alone . Escherichia coli was isolated in both patients . The presence of gas was confirmed early by ultrasound and CT scan of abdomen . Following treatment, good functional recovery was demonstrable in the affected kidneys by isotope renogram . We stress the need for early diagnosis of this condition and aggressive treatment with broad spectrum antibiotics. Proc Natl Sci Counc Repub China B, 1998 Jan, 22(1), 46 - 54 Beneficial effects of tetramethylpyrazine, an active constituent of Chinese herbs, on rats with endotoxemia; Liao MH et al.; Tetramethylpyrazine, an inhibitor of phosphodiesterase, has been widely used for treatment of cardiovascular diseases in China . Here, we investigate the effects of tetramethylpyrazine on hypotension, vascular hyporeactivity to norepinephrine (NE), release of tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO) in a rat model of circulatory shock induced by bacterial endotoxin (E . coli lipopolysaccharide, LPS) . Male Wistar-Kyoto rats were anesthetized and instrumented for the measurement of mean arterial pressure (MAP) and heart rate (HR) . Injection of LPS (10 mg/kg, i.v.) resulted in a fall in MAP and an increase of HR . In contrast, animals pretreated with tetramethylpyrazine (10 micrograms/kg, i.p . at 30 min prior to LPS) maintained a significantly higher MAP, but tachycardia was further enhanced at 60 min and 120 min when compared to rats given only LPS (LPS-rats) . The pressor effect of NE (1 microgram/kg, i.v.) was also significantly reduced after treatment of rats with LPS . Similarly, the thoracic aorta obtained from rats after in vivo studies showed a significant reduction in the contractile responses elicited by NE (1 microM) . Pretreatment of LPS-rats with tetramethylpyrazine partially, but significantly, prevented this LPS-induced hyporeactivity to NE in vivo and ex vivo . The injection of LPS resulted in a significant increase in the plasma TNF alpha level at 60 min, whereas the effect of LPS on the plasma nitrate (an indicator of NO formation) level increased in a time-dependent manner . This increment of both TNF alpha and nitrate levels induced by LPS was significantly reduced in LPS-rats pretreated with tetramethylpyrazine . The early hypotension caused by LPS was slightly, but significantly, prevented by pretreatment with tetramethylpyrazine, suggesting that tetramethylpyrazine affects the endothelial constitutive NOS (eNOS) . This was examined by the effect of tetramethylpyrazine on acetylcholine (ACh, 1 microM)-induced relaxation in rats treated with tetramethylpyrazine for 4 h . However, tetramethylpyrazine had no significant effects on the ACh-induced relaxation, indicating that tetramethylpyrazine does not affect the activity of eNOS . Thus, tetramethylpyrazine attenuates the early hypotension and the delayed circulatory failure caused by endotoxin in the rat . These effects may be due to inhibition of the release of circulation factors and TNF alpha, which usually reveal synergism upon the induction of iNOS. Mol Microbiol, 1998 Mar, 27(5), 1003 - 8 Modulations in lipid A and phospholipid biosynthesis pathways influence outer membrane protein assembly in Escherichia coli K-12; Kloser A et al.; The assembly defect of a mutant outer membrane protein, OmpF315, can be corrected by suppressor mutations that lower lipopolysaccharide (LPS) levels and indirectly elevate phospholipid levels . One such assembly suppressor mutation, asmB1, is an allele of lpxC (envA) whose product catalyses the first rate-limiting step in the lipid A (LPS) biosynthesis pathway . Besides reducing LPS levels, asmB1 confers sensitivity to MacConkey medium . A mutation, sabA1, that reverses the MacConkey sensitivity phenotype of asmB1 maps within fabZ (whose product is needed for phospholipid synthesis from a precursor) is also required for lipid A synthesis . In addition to reversing MacConkey sensitivity, the sabA1 mutation reverses the OmpF315 assembly suppression phenotype of asmB1 . These results show that OmpF315 assembly suppression by asmB1, which is achieved by lowering LPS levels, can be averted by a subsequent aberration in phospholipid synthesis at a point where the biosynthetic pathways for these two lipid molecules split . OmpF315 assembly suppression can also be achieved in an asmB+ background where FabZ expression is increased . The data obtained in this study provide genetic evidence that elevated phospholipid levels and/or phospholipid to LPS ratios are necessary for assembly suppression. Mol Microbiol, 1998 Mar, 27(5), 987 - 1001 An AUG initiation codon, not codon-anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli; Van Etten WJ et al.; We determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine-Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5'-AGGA-3' to 5'-UUUU-3' results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction . An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader . We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not . This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon-anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes . A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon-anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences . These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader. Mol Microbiol, 1998 Mar, 27(5), 875 - 87 Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C-terminal HPt domains of the sensor proteins; Perraud AL et al.; Despite the presence of highly conserved signalling modules, significant cross-communication between different two-component systems has only rarely been observed . Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two-component systems . Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively . One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS-TO-EvgS-R) was able to phosphorylate BvgA but not EvgA . In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS-T-EvgS-RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA . These results demonstrate that the C-terminal HPt domains of the sensor proteins endow the unorthodox two-component systems with a high specificity for the corresponding regulator protein . In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein. Microbiology, 1998 Mar, 144 ( Pt 3), 751 - 60 Expression of the second lysine decarboxylase gene of Escherichia coli; Lemonnier M et al.; Certain amino acids are substrates for two decarboxylase enzymes in Escherichia coli, one inducible by anaerobic growth at low pH and the other constitutive . In the case of lysine, an inducible decarboxylase (CadA) has been extensively characterized, but evidence for the existence of a second lysine decarboxylase is fragmentary and uncertain . This paper confirms that a second lysine decarboxylase is encoded by a locus (ldc) previously suggested to be a lysine decarboxylase gene on the basis of sequence comparisons . Overexpression of the cloned gene provided sufficient quantities of enzyme in cell-free extracts for preliminary examination of the properties of the ldc gene product, Ldc . The enzyme is active over a broad range of pH with an optimum at 7.6, much higher than that of CadA, about 5.5 . The temperature optimum for both enzymes is similar, at about 52 degrees C, but Ldc is more readily inactivated by heat than CadA . Expression of ldc from its own promoter was very weak for cells growing in a variety of media, although a low level of lysine decarboxylase was present in cells that carried the ldc region on an oligo-copy plasmid when these were grown in minimal-glucose medium . Northern analysis of RNA extracted from such cells revealed a transcript whose length corresponded to that of the ldc gene, suggesting that ldc is normally transcribed from a promoter immediately upstream . However, most of the ldc mRNA was shorter, indicating degradation or premature termination . The ldc upstream sequence promoted transcription of a lacZ gene to which it was fused . Introduction of the upstream sequence as an insert in a multicopy vector increased transcription of the resident lacZ fusion . The low level of expression in single copy, the emergence of expression when the gene is present at moderate copy number, and the derepression by the upstream sequence in trans imply that this second lysine decarboxylase gene may not be constitutive but subject to specific repression by a factor which remains to be identified. Microbiology, 1998 Mar, 144 ( Pt 3), 739 - 50 Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli; Kuzj AE et al.; The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course of Escherichia coli culture growth . mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle . The half-lives of cat mRNA and total mRNA also increase three- to fourfold during amino acid starvation when compared to exponential culture growth . Even though the stability of the cat message changes about fourfold during culture growth, the amount of cat mRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes in cat gene transcription . Translation of cat mRNA also changes during culture growth . In exponential phase, the rate of cat translation is about 14-fold higher than when the culture is in stationary phase . This is in contrast to the fourfold increase in stability of cat mRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability . When a stationary-phase culture was diluted into fresh medium, there was a five- to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin . These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited. Methods Enzymol, 1998, 290, 253 - 68 Structural analysis of GroE chaperonin complexes using chemical cross-linking; Azem A et al.; In this chapter, we have shown how chemical cross-linking with a bifunctional reagent, GA, can be used to investigate the structure of large oligomeric complexes such as GroEL14GroES7 and GroEL14(GroES7)2 . Cross-linking, followed by denaturing electrophoresis, confirmed the number and arrangement of GroEL and GroES subunits within each individual oligomer, which was previously known from EM analysis . Furthermore, cross-linking permitted a close examination of the effect of regulatory factors, such as nucleotides and free divalent cations, on the molecular structure of GroEL14, GroEL14GroES7, and GroEL14GroES7 . Finally, cross-linking analysis permitted characterization and quantitation of various chaperonin heterooligomeric complexes, GroEL14, GroEL14GroES7, and GroEL14GroES7 in solution, under conditions that also supported protein folding and ATP hydrolysis . It was shown that GA does not induce the artifactual association or the dissociation of GroES7 from the chaperonin . On the contrary, chemical cross-linking is an obligatory procedure when the subsequent analysis is carried out using methods that can displace the equilibrium. Mol Med, 1998 Feb, 4(2), 109 - 18 Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration; Gramzinski RA et al.; BACKGROUND: Attempts to optimize DNA vaccines in mice include using different routes of administration and different formulations . It may be more relevant to human use to carry such studies out in nonhuman primates . Here we compare different approaches to delivery of a DNA vaccine against the hepatitis B virus (HBV) in Aotus monkeys . MATERIALS AND METHODS: Thirty-two adult Aotus l . lemurinus monkeys divided into 8 groups of four were immunized with 400 microg of a DNA vaccine which encoded hepatitis B surface antigen (HBsAg) . DNA in saline was administered by intradermal (ID) or intramuscular (IM) injection with needle and syringe, IM injection with the Biojector needleless injection system or combined ID (needle) and IM (Biojector) . DNA formulated with cationic liposomes (CellFECTIN) was injected IM with needle or Biojector . DNA with added E . coli DNA (100 microg) was injected IM with the Biojector or ID . A ninth group of 4 monkeys was injected IM (needle) with Engerix-B, a commercial vaccine containing recombinant HBsAg (10 microg) adsorbed onto alum . Monkeys were boosted in an identical fashion to their prime at 8 weeks, but all received the protein vaccine (Engerix-B) at 16 weeks . Sera was assessed for antibodies against HBsAg (anti-HBs) by enzyme-linked imunosorbent assay (ELISA) . RESULTS: The primary humoral response induced by IM delivery of the DNA vaccine was very poor . In most cases there was no detectable anti-HBs even after 2 DNA doses but the kinetics of the response to subsequent protein indicated that a memory B cell response had been induced . In contrast, following IM-administration of DNA using the Biojector, detectable anti-HBs were observed in 3 of 8 animals and evidence for immunological priming was apparent in an additional 4 of the 8 monkeys . ID injection of DNA vaccine in saline induced a potent antibody response which was augmented 6-fold by the addition of E . coli DNA . Combining ID and IM administration did not improve humoral immunity over ID injection alone . CONCLUSIONS: For immunization of primates with DNA vaccines, ID may be a preferable route to IM, although it is not clear whether the Aotus monkey is a relevant model for humans in this respect . Nevertheless, the use of the Biojector needleless injection system may improve responses with IM delivery of DNA vaccines . As well, the immunostimulatory action of E . coli DNA may be used to augment the humoral response induced by a DNA vaccine. Science, 1998 Mar 20, 279(5358), 1958 - 61 Redesigning enzyme topology by directed evolution; MacBeath G et al.; Genetic selection was exploited in combination with structure-based design to transform an intimately entwined, dimeric chorismate mutase into a monomeric, four-helix-bundle protein with near native activity . Successful reengineering depended on choosing a thermostable starting protein, introducing point mutations that preferentially destabilize the wild-type dimer, and using directed evolution to optimize an inserted interhelical turn . Contrary to expectations based on studies of other four-helix-bundle proteins, only a small fraction of possible turn sequences (fewer than 0.05 percent) yielded well-behaved, monomeric, and highly active enzymes . Selection for catalytic function thus provides an efficient yet stringent method for rapidly assessing correctly folded polypeptides and may prove generally useful for protein design. J Biol Chem, 1998 Mar 13, 273(11), 6395 - 401 The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease . Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity; Huang HC et al.; Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R . catesbeiana (bullfrog) oocytes . It possesses both ribonuclease activity and cytotoxicity against tumor cells . We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored . An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers . Full-length cDNA was obtained by 5'- and 3'-RACE technique . The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site . The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3) . The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity . The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes . Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity . Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity . DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes . This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily. J Biol Chem, 1998 Mar 13, 273(11), 6303 - 11 Characterization of a mammalian peroxiredoxin that contains one conserved cysteine; Kang SW et al.; A new type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces H2O2 with the use of electrons from thioredoxin and contains two essential cysteines was recently identified . TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine . Recombinant human 1-Cys Prx expressed in and purified from Escherichia coli has now been shown to reduce H2O2 with electrons provided by dithiothreitol . Furthermore, human 1-Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response either to the addition of exogenous H2O2 or to treatment with platelet-derived growth factor . The conserved Cys47-SH group was shown to be the site of oxidation by H2O2 . Thus, mutation of Cys47 to serine abolished peroxidase activity . Moreover, the oxidized intermediate appears to be Cys-SOH . In contrast to TPx, in which one of the two conserved cysteines is oxidized to Cys-SOH and then immediately reacts with the second conserved cysteine of the second subunit of the enzyme homodimer to form an intermolecular disulfide, the Cys-SOH of 1-Cys Prx does not form a disulfide . Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys-SeOH of oxidized glutathione peroxidase, was able to reduce the Cys-SOH of 1-Cys Prx and consequently could not support peroxidase activity . Human 1-Cys Prx was previously shown to exhibit a low level of phospholipase A2 activity at an acidic pH; the enzyme was thus proposed to be lysosomal, and Ser32 was proposed to be critical for lipase function . However, the mutation of Ser32 or Cys47 has now been shown to have no effect on the lipase activity of 1-Cys Prx, which was also shown to be a cytosolic protein . Thus, the primary cellular function of 1-Cys Prx appears to be to reduce peroxides with the use of electrons provided by an as yet unidentified source; the enzyme therefore represents a new type of peroxidase. J Biol Chem, 1998 Mar 13, 273(11), 6248 - 53 Chorismate mutase-prephenate dehydratase from Escherichia coli . Study of catalytic and regulatory domains using genetically engineered proteins; Zhang S et al.; The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine) . Six genes coding for P-protein domains or subdomains were constructed and successfully expressed . Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition . Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity . Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding . The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein . Both the mutase domain and the native P-protein formed dimers . Deletion of the mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects . Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase for chorismate. J Biol Chem, 1998 Mar 13, 273(11), 6242 - 7 Localization and functional analysis of the substrate specificity/catalytic domains of human M-form and P-form phenol sulfotransferases; Sakakibara Y et al.; Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties . A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs . Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84-148 contain the structural determinants for the substrate specificity of either M-form or P-form PST . Data on the kinetic constants (Km, Vmax, and Vmax/Km) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84-89 and Region II spanning amino acid residues 143-148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol . In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST . Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs . Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding of D-p-tyrosine to wild-type M-form PST . Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84-148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties. J Biol Chem, 1998 Mar 13, 273(11), 6149 - 56 NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax . The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties; Brunner NA et al.; The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction . NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate . The coding gene was cloned, sequenced, and expressed in Escherichia coli . Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases . The activity of the enzyme is affected by a series of metabolites . All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+ . Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+ . Additionally, most of the effectors investigated induce cooperativity of NAD+ binding . The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T . tenax . Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway. J Biol Chem, 1998 Mar 13, 273(11), 6066 - 73 Serine hydroxymethyltransferase is maternally essential in Caenorhabditis elegans; Vatcher GP et al.; The mel-32 gene in the free living soil nematode Caenorhabditis elegans encodes a serine hydroxymethyltransferase (SHMT) isoform . Seventeen ethylmethanesulfonate (EMS)-induced mutant alleles of mel-32(SHMT) have been generated, each of which causes a recessive maternal effect lethal phenotype . Animals homozygous for the SHMT mutations have no observable mutant phenotype, but their offspring display an embryonic lethal phenotype . The Mel-32 phenotype has been rescued with a transgenic array containing only mel-32(SHMT) genomic DNA . Heteroduplex analysis of the 17 alleles allowed 14 of the mutations to be positioned to small regions . Subsequent sequence analysis has shown that 16 of the alleles alter highly conserved amino acids, while one allele introduces a stop codon that truncates two thirds of the predicted protein . mel-32(SHMT) has a 55-60% identity at the amino acid level with both isoforms of SHMT found in yeast and humans and a 50% identity with the Escherichia coli isoform . The C . elegans mel-32 mutation represents the first case where SHMT has been shown to be an essential gene. J Biol Chem, 1998 Mar 13, 273(11), 6050 - 6 Identification of active site residues in Escherichia coli DNA topoisomerase I; Chen SJ et al.; Alanine substitution mutagenesis of Escherichia coli DNA topoisomerase I, a member of the type IA subfamily of DNA topoisomerases, was carried out to identify amino acid side chains that are involved in transesterification between DNA and the active site tyrosine Tyr-319 of the enzyme . Twelve polar residues that are highly conserved among the type IA enzymes, Glu-9, His-33, Asp-111, Glu-115, Gln-309, Glu-313, Thr-318, Arg-321, Thr-322, Asp-323, His-365, and Thr-496, were selected for alanine substitution . Each of the mutant enzymes was overexpressed, purified, and characterized . Surprisingly, only substitution at Glu-9 and Arg-321 was found to reduce the DNA relaxation activity of the enzyme to an insignificant level . The R321A mutant enzyme, but not the E9A mutant enzyme, was found to retain a reduced level of DNA cleavage activity . Two additional mutant enzymes R321K and E9Q were also constructed and purified . Replacing Arg-321 by lysine has little effect on enzymatic activities; replacing Glu-9 by glutamine greatly reduces the supercoil removal activity but not the DNA cleavage and rejoining activities . From these results and the locations of the amino acids in the crystal structure of the enzyme, it appears that Glu-9 has a critical role in DNA breakage and rejoining, probably through its interaction with the 3' deoxyribosyl oxygen . The positively charged Arg-321 may also participate in these reactions by interacting with the scissile DNA phosphate as a monodentate . Because of the strict conservation of these residues, the findings for the E . coli enzyme are likely to apply to all type IA DNA topoisomerases. J Biol Chem, 1998 Mar 13, 273(11), 6024 - 9 Biochemical and phylogenetic characterization of the dUTPase from the archaeal virus SIRV; Prangishvili D et al.; The derived amino acid sequence from a 474-base pair open reading frame in the genome of the Sulfolobus islandicus rod-shaped virus SIRV shows striking similarity to bacterial dCTP deaminases and to dUTPases from eukaryotes, bacteria, Poxviridae, and Retroviridae . The putative gene was expressed in Escherichia coli, and dUTPase activity of the recombinant enzyme was demonstrated by hydrolysis of dUTP to dUMP . Deamination of dCTP by the enzyme was not detected . Phylogenetic analysis based on amino acid sequences of the characterized enzyme and its homologues showed that the dUTPase-encoding dut genes and the dCTP deaminase-encoding dcd genes constitute a paralogous gene family . This report is the first identification and functional characterization of an archaeal dUTPase and the first phylogeny derived for the dcd-dut gene family. J Biol Chem, 1998 Mar 6, 273(10), 5655 - 61 Role of the dimeric structure in Cu,Zn superoxide dismutase . pH-dependent, reversible denaturation of the monomeric enzyme from Escherichia coli; Battistoni A et al.; To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated . Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein . Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface . The melting temperature of the E . coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66 degrees C at pH 7.8 and at 79.3 degrees C at pH 6.0 . The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources . Apo-superoxide dismutase from E . coli was shown to be nearly as stable as the bovine apoenzyme, whose holo form is much more stable and less sensitive to pH variations . The remarkable pH susceptibility of the E . coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes . Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E . coli enzyme was shown to be reversibly perturbed by urea . These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the beta-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E . coli enzyme are due to higher flexibility and accessibility to the solvent of its active-site region. J Biol Chem, 1998 Mar 6, 273(10), 5520 - 7 The mutagenesis protein MucB interacts with single strand DNA binding protein and induces a major conformational change in its complex with single-stranded DNA; Sarov-Blat L et al.; The MucA and MucB proteins are plasmid-encoded homologues of the Escherichia coli UmuD and UmuC proteins, respectively . These proteins are required for SOS mutagenesis, although their mechanism of action is unknown . By using the yeast two-hybrid system we have discovered that MucB interacts with SSB, the single strand DNA binding protein (SSB) of E . coli . To examine the interaction at the protein level, the MucA, MucA', and MucB proteins were overproduced, purified in denatured state, and refolded . Purified MucA and MucA' each formed homodimers, whereas MucB was a monomer under native conditions . RecA promoted the cleavage of MucA to MucA', and MucB was found to bind single-stranded DNA (ssDNA), similarly to the properties of the homologous UmuD and UmuC proteins . Purified MucB caused a shift in the migration of SSB in a sucrose density gradient, consistent with an interaction between these proteins . Addition of MucB to SSB-coated ssDNA caused increased electrophoretic mobility of the nucleoprotein complex and increased staining of the DNA by ethidium bromide . Analysis of radiolabeled SSB in the complexes revealed that only a marginal release of SSB occurred upon addition of MucB . These results suggest that MucB induces a major conformational change in the SSB.ssDNA complex but does not promote massive release of SSB from the DNA . The interaction with SSB might be related to the role of MucB in SOS-regulated mutagenesis. Biochemistry, 1998 Mar 3, 37(9), 3210 - 9 Coupling of site-specific DNA binding to protein dimerization in assembly of the biotin repressor-biotin operator complex; Streaker ED et al.; The Escherichia coli repressor of biotin biosynthesis, BirA, binds site-specifically to the biotin operator, a 40 base pair imperfect inverted palindrome . Two repressor monomers have been shown to bind to the two operator half-sites . Analysis of results of quantitative DNase I footprint titrations performed on the wild-type biotin operator template indicate that binding is well described by a cooperative mechanism . The data obtained from these studies were, however, insufficient to independently resolve all of the energetic parameters associated with cooperative binding of the two repressor monomers to the operator site . In this work, to further dissect the energetics of assembly of the biotin repressor-biotin operator complex, measurements of binding of BirA to four bioO variants designed to reduce the valency of repressor binding from 2 to 1 have been performed . Results of these measurements indicate, as was found with the wild-type biotin operator template, that two repressor monomers bind simultaneously to the two half-sites of all variant operators . Protein dimerization and DNA binding are thus obligatorily coupled in the biotin repressor system . Furthermore, the results suggest that, in the context of a cooperative binding mechanism, the cooperative free energy associated with the biotin repressor-biotin operator interaction is significantly more favorable than the previously estimated -2 kcal/mol. Biochemistry, 1998 Mar 3, 37(9), 3187 - 95 Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding; Yuan T et al.; The two interaction surfaces of the dumbbell-shaped calcium-regulatory protein calmodulin (CaM) are rich in the amino acid Met . In this work we have used fluorescence spectroscopy to study the role of these Met residues in binding the single Trp residue that is found in many CaM-binding domain peptides . This approach is facilitated by the absence of Trp residues in CaM . In addition to the wild-type protein, we studied CaM containing the unnatural amino acid selenomethionine (SeMet), which was biosynthetically substituted for its nine Met residues . Furthermore, a CaM mutant protein in which all four Met residues in the C-terminal domain were mutated to Leu, and the N-terminal domain contained either Met or the unnatural SeMet, was studied . The Trp fluorescence quantum yield of many Trp-containing CaM-binding peptides increases upon binding to calcium-CaM . Moreover, the emission wavelength of the Trp fluorescence is blue-shifted from 353 to 325-333 nm . These parameters indicate movement of Trp from a solvent exposed to a hydrophobic environment . The fluorescence results obtained with these four CaM variants showed that Se is very effective at quenching Trp fluorescence in the calmodulin-bound peptides from myosin light chain kinase (MLCK) and CaM kinase I, while S is somewhat effective (Se > S > C) . The quenching effect is markedly distance dependent, as it only influences the Trp residue of the bound peptide (<=7 A) but has little effect on the two Tyr residues in the C-terminal domain of CaM (>=10 A) . Since the Trp fluorescence quenching is very dramatic, the protein containing Leu's in the C-terminal domain and SeMet's in the N-terminal domain allowed us to directly determine the orientation of the MLCK and CaM kinase I peptides bound to CaM; in both cases the Trp residue binds to the C-terminal domain of CaM . Our data indicate that SeMet quenching of Trp fluorescence could become a simple and useful tool for studies of protein folding, and protein-protein and protein-peptide interactions. Biochemistry, 1998 Mar 3, 37(9), 3116 - 36 Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure; Jezewska MJ et al.; Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods . DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork . Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm . The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm . Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork . The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm . In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm . This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork . The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm . The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E . coli DNA. Biochemistry, 1998 Mar 3, 37(9), 3068 - 77 Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase; Piersma SR et al.; The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm . Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde . The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325 nm . Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible . The fluorescence emission spectrum (excitation at 325 nm) for NADH bound to the enzyme has a maximum at 422 nm . The fluorescence intensity is enhanced by a factor of 3 upon binding of isobutyramide (Kd = 59 microM) . Isobutyramide acts as competitive inhibitor (Ki = 46 microM) with respect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor . The nonreactive substrate analogue trifluoroethanol acts as a competitive inhibitor with respect to the substrate ethanol (Ki = 1.6 microM), which binds to the enzyme containing the oxidized cofactor . Far-UV circular dichroism spectra of the enzyme containing NADH and the enzyme containing NAD+ were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor . Near-UV circular dichroism spectra of NADH bound to the enzyme have a minimum at 323 nm (Deltaepsilon = -8.6 M-1 cm-1) . The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor . This implies a rigid environment as well as lack of motion of the fluorophore . The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degreesC and could be resolved into at least three components (in the range 0.13-0.96 ns) . Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state lifetime increased to 1.02 ns and could be resolved into two components (0.37 and 1.11 ns) . The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima compared to other NADH-dehydrogenase complexes, but comparable to that observed for NADH bound to horse liver alcohol dehydrogenase . The fluorescence lifetime of NADH bound to the nicotinoprotein is very short compared to enzyme-bound NADH complexes, also compared to NADH bound to horse liver alcohol dehydrogenase . The cofactor-protein interaction in the nicotinoprotein alcohol dehydrogenase active site is more rigid and apolar than that in horse liver alcohol dehydrogenase . The optical properties of NADH bound to nicotinoprotein alcohol dehydrogenase differ considerably from NADH (tightly) bound to UDP-galactose epimerase from Escherichia coli . This indicates that although both enzymes have NAD(H) as nonexchangeable cofactor, the NADH binding sites are quite different. Biochemistry, 1998 Mar 3, 37(9), 3043 - 52 Analysis of the pH- and ligand-induced spectral transitions of tryptophanase: activation of the coenzyme at the early steps of the catalytic cycle; Ikushiro H et al.; Tryptophanase has an absorption maximum at 338 nm at high pH and 422 nm at low pH . The 422-nm absorption species has been considered to be the catalytically competent ketoenamine form of the Schiff base of pyridoxal 5'-phosphate with a lysine residue . The 338-nm absorption band showed an intense fluorescence band at 390 nm and not around 500 nm, indicating that the 338-nm absorption species is the substituted aldamine rather than an enolimine form of the Schiff base which has been suggested previously . To explore the mechanism of the enzyme that can exert its catalytic ability at high pH where most of its coenzyme exists as the catalytically incompetent aldamine structure, the reaction of tryptophanase with 3-indolepropionate, a substrate analogue that stops the reaction at the step of the Michaelis complex, was studied at various pH values and analogue concentrations . Kinetic analysis was done based on a scheme involving eight forms of the enzyme, i.e., the liganded and unliganded forms of the ketoenamine, the substituted aldamine structures, and their protonated and deprotonated forms . Kinetic parameters were obtained for each interconversion step . The results showed that the binding of 3-indolepropionate to tryptophanase shifts the equilibrium from the substituted aldamine to the ketoenamine structure over the entire pH region studied . This implies that in the reaction of tryptophanase with tryptophan at high pH, where the enzyme shows maximum activity, the binding of the substrate to the enzyme converts the inactive aldamine form of the coenzyme to the active ketoenamine form . Mechanisms for the activation process, in which a nucleophile is expelled from the aldamine either by steric hindrance of the nucleophile with the ligand or by the negative charge of the ligand alpha-carboxylate group that stabilizes the aldimine structure, were discussed. Biochemistry, 1998 Mar 3, 37(9), 2979 - 90 Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment; Furey WS et al.; Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I . Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors to the donor fluorescein fluorophore used to modify cysteine 751 in the double mutant KF (S751C, C907S) . Design of the template-primer allowed the probe's position within the DNA-protein complex to be varied by stepwise extension of the primer strand upon addition of the appropriate deoxynucleoside triphosphates (dNTP) . The TMR acceptor probe occupied seven different positions in the template-primers, five in the single stranded region and two in the double stranded region . The efficiency of energy transfer was determined at each position by calculating the integrated area of the fluorescein emission peak in the presence and absence of acceptor . Results indicate that the FRET efficiency varied in a sinusoidal fashion with a periodicity of approximately 10 base pairs and that the data could be fitted to an equation derived from a simple model formulated on the basis of helical structure . The data support the conclusion that the single stranded template portion of a DNA template-primer adopts a helical conformation when bound to the KF . The results of this study further support FRET as a useful method for the determination of structure and conformation in protein-DNA complexes. Biochemistry, 1998 Mar 3, 37(9), 2949 - 60 Interfacial communications in recombinant rabbit kidney pyruvate kinase; Friesen RH et al.; Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery . The muscle- and kidney-type isozymes are coded by the same gene . As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues . However, they exhibit very different regulatory behavior . In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia coli . The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure . Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg . This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors . The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator . In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor . These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system . First, all substrates and effectors communicate through this particular dimer-dimer interface . Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe. Biochemistry, 1998 Mar 3, 37(9), 2941 - 8 Interaction of the hydrogenase accessory protein HypC with HycE, the large subunit of Escherichia coli hydrogenase 3 during enzyme maturation; Drapal N et al.; Maturation of the large subunit of E . coli hydrogenase 3, HycE, requires the action of seven accessory proteins . The HycI protease catalyses a C-terminal proteolytic cleavage of the large subunit, which was shown to result in a dramatic change in migration behavior of HycE in nondenaturing PAGE . HypA, HypB, HypC, HypD, HypE, and HypF are required for metallocenter assembly . A polyacrylamide gel system under nondenaturing conditions was used for the investigation of any protein-protein interactions between HycE and the Hyp proteins . It revealed the existence of a complex between the precursor of HycE (pre-HycE) with one of the accessory proteins, namely HypC . HypC migrates in at least three different forms in nondenaturing PAGE, the appearance of one of which (form 1) is strictly dependent on the presence of unprocessed HycE in the extract . Overexpression of either hypC or hycE from a plasmid leads to an increased formation of this HypC-form 1 . In two-dimensional polyacrylamide gel electrophoresis with nondenaturing PAGE as the first and SDS-PAGE as the second dimension, this HypC form comigrates with part of the pre-HycE protein . This comigration was also observed in anion exchange chromatography . To analyze the pre-HycE-HypC complex in more detail, HypC was overproduced and purified . The purified protein was able to bind to pre-HycE in vitro . These results and also the finding that the processed form of HycE is not associated with HypC suggest that HypC binds to pre-HycE to keep it in a conformation accessible for metal incorporation. Biochemistry, 1998 Mar 3, 37(9), 2905 - 11 Semifunctional site-specific mutants affecting the hydrolytic half-reaction of microsomal epoxide hydrolase; Tzeng HF et al.; Microsomal epoxide hydrolase (MEH) is a member of the alpha/beta-hydrolase fold family of enzymes, each of which has a catalytic triad consisting of a nucleophile involved in the formation of a covalent intermediate and a general base and charge relay carboxylate that catalyze the hydrolysis of the intermediate . The rate-limiting step in the catalytic mechanism of MEH is hydrolysis of the ester intermediate . An efficient bacterial expression system for a C-terminal hexahistidine tagged version of the native enzyme, which facilitates the isolation of mutant enzymes in which residues involved in the hydrolytic half-reaction have been altered, is described . The H431S mutant of this enzyme is efficiently alkylated by substrate to form the ester intermediate but is unable to hydrolyze the ester to complete the catalytic cycle, a fact that confirms that H431 acts as the base in the hydrolytic half-reaction . The charge relay carboxylate, which is not apparent in paired sequence alignments with other alpha/beta-hydrolase fold enzymes, is thought to be located between residues 340 and 405 . A mutagenic survey of all eight Asp and Glu residues in this region reveals that only two (E376 and E404) influence the catalytic mechanism . Steady-state and pre-steady-state kinetic analyses of these residues suggest that both E404 and E376 may serve the charge relay function in the hydrolysis half-reaction . Finally, the tryptophan residue (W150), which resides in the oxyanion hole sequence HGWP, is demonstrated to contribute to the large change in intrinsic protein fluorescence observed when the enzyme is alkylated. Biochemistry, 1998 Mar 3, 37(9), 2897 - 904 Mechanism of microsomal epoxide hydrolase . Semifunctional site-specific mutants affecting the alkylation half-reaction; Laughlin LT et al.; Microsomal epoxide hydrolase (MEH) catalyzes the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester . An efficient bacterial expression system for the enzyme from rat that facilitates the production of native and mutant enzymes for mechanistic analysis is described . Pre-steady-state kinetics of the native enzyme toward glycidyl-4-nitrobenzoates, 1, indicate the rate-limiting step in the reaction is hydrolysis of the alkyl-enzyme intermediate . The enzyme is enantioselective, turning over (2R)-1 about 10-fold more efficiently than (2S)-1, and regiospecific toward both substrates with exclusive attack at the least hindered oxirane carbon . Facile isomerization of the monoglyceride product is observed and complicates the regiochemical analysis . The D226E and D226N mutants of the protein are catalytically inactive, behavior that is consistent with the role of D226 as the active-site nucleophile as suggested by sequence alignments with other alpha/beta-hydrolase fold enzymes . The D226N mutant undergoes hydrolytic autoactivation with a half-life of 9.3 days at 37 degreesC, suggesting that the mutant is still capable of catalyzing the hydrolytic half-reaction (in this instance an amidase reaction) and confirming that D226 is in the active site . The indoylyl side chain of W227, which is in or near the active site, is not required for efficient alkylation of the enzyme or for hydrolysis of the intermediate . However, the W227F mutant does exhibit altered stereoselectivity toward (2R)-1, (2S)-1, and phenanthrene-9,10-oxide, suggesting that modifications at this position might be used to manipulate the stereo- and regioselectivity of the enzyme. Biochemistry, 1998 Mar 3, 37(9), 2800 - 6 Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase); Yamazaki T et al.; Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin . The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum . In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone . The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione . The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme . The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone . The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent fur