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| Zentralbl Bakteriol {Orig B}, 1976 Jul, 162(1-2), 180 - 3 {Microbial breakdown of caffeine (author's transl)}; Blecher R; A bacterium, capable of growing aerobically with caffeine as its sole source of carbon and nitrogen, was isolated from soil and identified as Pseudomonas putida sp . The breakdown of caffeine begins with stepwise demethylation, which leads via various N-methyl-purines to xanthine each step yielding formaldehyde . Xanthine is then broken down via uric acid, allantoin, allantoic acid and further intermediates to urea and glyoxylic acid, which serves as the actual source of carbon. J Biochem (Tokyo), 1976 Jun, 79(6), 1381 - 3 Purification, crystallization, and some properties of creatine amidinohydrolase from Pseudomonas putida; Yoshimoto T et al.; A method was developed for purification and crystallization of creatinase {creatine amidinohydrolase, EC 3.5.3.3} from Pseudomonas putida var . naraensis C-83 . The purified preparation appeared homogeneous on disc electrophoresis and ultracentrifugation and had a molecular weight of 94,000 . It was most active at pH 8 and stable between pH 6 and 8 for 24 hr at 37 degrees . SDS-polyacrylamide gel electrophoresis indicated that the native enzyme was made up of two subunit monomers, the molecular weights of which were estimated to be 47,000 . Inhibition experiments suggested that a sulfhydryl group is located in or near the active site of the enzyme. J Biochem (Tokyo), 1976 Jun, 79(6), 1263 - 72 Purification and characterization of methioninase from Pseudomonas putida; Ito S et al.; Methioninase of Pseudomonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract . 1 . The purified enzyme had an S20,w of 8.37, a molecular weight of 160,000, and an isoelectric point of 5.6 . 2 . A break in the Arrhenius plot was observed at 40 degrees and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively . 3 . In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates . D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert . Equimolar formation of alpha-ketobutyrate and CH3SH was observed with methionine as a substrate . 4 . In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5 . Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks . Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 muM . 5 . The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating). J Bacteriol, 1976 May, 126(2), 794 - 8 Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida; Benson S et al.; We have identified an alcohol dehydrogenase activity in Pseudomonas putida strains carrying the CAM-OCT degradative plasmid that were grown on octane . The activity is nicotinamide adenine dinucleotide independent, sediments at 48,000 x g, and shows 20-fold greater activity with octanol rather than butanol as substrate . The enzyme is inducible by unoxidized alkane and is present only in strains that have the OCT plasmid genes for alkane degradation with a wild-type alcO locus . No analogous chromosomal dehydrogenase could be detected . Wild-type and actanol-negative mutants (alcA-) without plasmids both contain a constitutive nicotinamide adenine dinucleotide-linked soluble alcohol dehydrogenase activity . This means that alcA- mutants are cryptic for octanol oxidation and suggests that the particulate plasmid-coded alcohol dehydrogenase activity is active on surface- or membrane-bound substrate. J Bacteriol, 1976 Apr, 126(1), 272 - 81 Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida; Parke D et al.; Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source . The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system . The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate . The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains . Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes . Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively . Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type . Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems. J Gen Microbiol, 1976 Apr, 93(2), 227 - 40 Failure of complex supplementation of minimal cultures to elicit a shift-up response in Pseudomonas putida; Collins JK et al.; The addition of complex supplements (particularly amino acids) to cultures of Pseudomonas putida growing on a good carbon source did not result in a substantial increase in the growth rate . Amino acids entered the cells within 30 s of addition and reached significant internal pool concentrations . Endogenous amino acid biosynthesis was quickly inhibited (about 75%), with a substantial sparing of the original carbon source . Within 20 min of supplementation significant respiration of added amino acids was detected, yet the ATP pool size did not increase and the bacteria did not grow faster . The RNA content of P . putida growing in complex medium differed from that of enteric bacteria in that, although it varied with growth rate, it was not substantially larger than the RNA content of bacteria grown in a minimal medium with a good carbon and energy source . The rate of RNA accumulation on shift-up remained substantially unchanged on supplementation if the minimal medium had a carbon source producing fast growth, and did not increase for about 30 min if the carbon source was relatively poor . In other respects RNA synthesis was similar to that of the enteric bacteria, being stringently controlled, inhibited by trimethoprim and continuing in the presence of chloramphenicol . It is proposed that growth of P . putida in complex media is limited by the rate of synthesis of stable RNA. J Bacteriol, 1976 Apr, 126(1), 410 - 6 Isolation of plasmid deoxyribonucleic acid from Pseudomonas putida; Palchaudhuri S et al.; Conditions suitable for reproducible recovery of covalently closed circular deoxyribonucleic acid from strains of Pseudomonas putida containing degradative plasmids (CAM, SAL, OCT, etc.) have been defined . These degradative plasmids could not be isolated by the usual procedure, whereas RP1, an R factor of the P group, present in the isogenic strain of P . putida, was isolated equally well by either the usual procedure or the modified procedure . Characterization by electron microscopy of RP1 deoxyribonucleic acid confirmed the molecular weight (about 40 X 10(6)) previously determined by sucrose gradient centrifugation. Mol Gen Genet, 1976 Mar 30, 144(3), 307 - 11 Supra-operonic clustering of genes specifying glucose dissimilation in Pseudomonas putida; de Torrontegui G et al.; The linkage arrangements of genes governing glucolysis in Pseudomonas putida have been determined by transductional analysis . Five genes (gdh, kgtA, kgtB, edd and eda), comprising at least three operons, are contransducible with each other, but not with ggu (glucose and gluconate uptake) nor with genes of a known supra-operonic cluster of genes specifying enzymes of other dissimilatory pathways, nor with a biochemically uncharacterized his marker . It thus appears that P . putida may have more than one chromosomal region in which genes with dissimilatory function are clustered in a supro-operonic fashion. J Biol Chem, 1976 Mar 10, 251(5), 1385 - 91 Multiple forms of rat liver cytochrome P-450 . Immunochemical evidence with antibody against cytochrome P-448; Thomas PE et al.; Purified hepatic cytochrome P-448 from 3-methylcholanthrene-treated rats was used to produce antibody in rabbits . The cytochrome P-448 antibody (IgG fraction) isolated from immune rabbit serum is quite specific and precipitates purified rat liver cytochrome P-448 at low antibody to protein ratios when assayed by the Ouchterlony double diffusion technique . Purified hepatic cytochrome P-450 from phenobarbital-treated rats cross-reacts poorly with the cytochrome P-448 antibody as do purified rabbit hepatic cytochrome P-448 and P-450 . No cross-reaction is observed with purified cytochrome P-450 from beef adrenal mitochondria or from Pseudomonas putida in Ouchterlony double diffusion experiments . The cytochrome P-448 antibody produces a single distinct precipitin band with purified rat cytochrome P-448 . In contrast, purified liver cytochrome P-450 from phenobarbital-treated rats gives three precipitin bands, all of which contain hemeprotein as judged by benzidine staining . At least two of the three precipitin bands are immunochemically different from the precipitin band formed with cytochrome P-448 . When added to the reconstituted system, the cytochrome P-448 antibody inhibits purified rat cytochrome P-448- and P-450-supported N-demethylation of benzphetamine, O-deethylation of ethoxycoumarin, hydroxylation of benzo{a}pyrene, and the hydroxylation of testosterone at the 6beta, 7alpha, and 16alpha positions . Antibody inhibits cytochrome P-448-supported metabolism more than cytochrome P-450-supported metabolism except for benzo{a}pyrene hydroxylation at low antibody to hemeprotein ratios . In addition, the pattern and extent of inhibition of the cytochrome P-450 system depends on the substrate used, suggesting that multiple forms of the hemeprotein are present in the purified preparation from phenobarbital-treated rats . The observed patterns of immunoprecipitation and inhibition of catalytic activity indicate that (a) cytochrome P-448 from 3-methylcholanthrene-treated rats is immunochemically different from cytochrome P-450 from phenobarbital-treated rats, and (b) there appear to be at least three hemeprotein forms in the purified cytochrome P-450 preparation from phenobarbital-treated rats. J Bacteriol, 1976 Mar, 125(3), 975 - 84 Metabolism of resorcinylic compounds by bacteria: orcinol pathway in Pseudomonas putida; Chapman PJ et al.; Enrichment cultures yielded two strains of Pseudomonas putida capable of growth with orcinol (3,5-dihydroxytoluene) as the sole source of carbon . Experiments with cell suspensions and cell extracts indicate that orcinol is metabolized by hydroxylation of the benzene ring followed successively by ring cleavage and hydrolyses to give 2 mol of acetate and 1 mol of pyruvate per mol of orcinol as shown: orcinol leads to 2,3,5-trihydroxytoluene leads to 2,4,6-trioxoheptanoate leads to acetate + acetylpyruvate leads to acetate + pyruvate . Evidence for this pathway is based on: (i) high respiratory activities of orcinol-grown cells towards 2,3,5-trihydroxytoluene; (ii) transient accumulation of a quinone, probably 2-hydroxy-6-methyl-1,4-benzoquinone, during grouth with orcinol; (iii) formation of pyruvate and acetate from orcinol, 2,3,5-trihydroxytoluene, and acetylpyruvate catalyzed by extracts of orcinol, but not by succinate-grown cells; (iv) characterization of the product of oxidation of 3-methylcatechol (an analogue of 2,3,5-trihydroxytoluene) showing that oxygenative cleavage occurs between carbons bearing methyl and hydroxyl substituents; (v) transient appearance of a compound having spectral properties similar to those of acetylpyruvate during 2,3,5-trihydroxytoluene oxidation by extracts of orcinol-grown cells . Orcinol hydroxylase exhibits catalytic activity when resorcinol or m-cresol is substituted for orcinol; hydroxyquinol and 3-methylcatechol are substrates for the ring cleavage enzyme 2,3,5-trihydroxytoluene-1,2-oxygenase . The enzymes of this pathway are induced by growth with orcinol but not with glucose or succinate. J Bacteriol, 1976 Mar, 125(3), 818 - 28 Ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new TOL plasmids; Williams PA et al.; Thirteen bacteria have been isolated from nine different soil samples by selective enrichment culture on m-toluate (m-methylbenzoate) minimal medium . Eight of these were classified as Pseudomonas putida, one as a fluorescent Pseudomonas sp., and four as nonfluorescent Pseudomonas sp . All 13 strains appeared to carry TOL plasmids superficially similar to that previously described in P . putida mt-2 in that: (i) all the wild-type strains could utilize toluene, m-xylene, and p-xylene as sole carbon and energy sources, (ii) these growth substrates were metabolized through the corresponding alcohols and aldehydes to benzoate, m-toluate, and p-toluate, respectively, and thence by the divergent meta (or alpha-ketoacid) pathway, and (iii) the isolates could simultaneously and spontaneously lose their ability to utilize the hydrocarbons, alcohols, aldehydes, and acids, particularly during growth on benzoate, giving rise to cured strains which could grow only on benzaldehyde and benzoate of the aromatic substrates by the alternative ortho (or beta-ketoadipate) pathway . Eight of the isolates were able to transfer their TOL plasmids into their own cured strains, but only five were able to transfer them in interstrain conjugation into the cured strains, but only five were able to transfer them in interstrain conjugation into the cured derivative of P . putida mt-2 . However, P . putida mt-2 was able to transfer its TOL plasmid into 11 of the cured isolates, and eight of these were able to retransmit this foreign plasmid in intrastrain conjugation with their own cured derivatives . Three of the isolates, MT 14, MT 15, and MT 20, differed significantly from the others in that the wild-type strains dissimilated the p-methyl-substituted substrates poorly, and also, during growth on benzoate, in addition to the cured derivatives, they gave rise to derivatives with a phenotype intermediate between the cured and wild-type strains, the biochemical and genetic nature of which has not been elucidated. J Bacteriol, 1976 Mar, 125(3), 985 - 98 Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida; Chapman PJ et al.; Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol . Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol . One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway . After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase . Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate . Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction . P . putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates . The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described . Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P . putida ORC grown with resorcinol . Its further metabolism was reduced nicotinamide adenine dinucleotide dependent. J Biol Chem, 1976 Feb 25, 251(4), 941 - 9 Properties of anthranilate synthetase component II from Pseudomonas putida; Goto Y et al.; The interaction of Pseudomanas putida anthranilate synthetase Component II (AS II) with glutamine, glutamine analogs, and iodoacetamide has been investigated in order to clarify the initial steps in the mechanism for glutamine utilization . AS II is alkylated and irreversibly inactivated by covalent attachment of approximately 1 eg of L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone) or 1 eq of iodoacetamide . Alkylation of AS II by chloroketone involves initial formation of an enzyme-inhibitor complex having a Ki of 28 muM . Alkylation of AS II by iodoacetamide occurs without initial formation of a reversible complex . In both cases glutamine protects against alkylation and exhibits competitive kinetics . When anthranilate synthetase Component I (AS I) is associated with AS II, the second substrate, chorismate, enhances alkylation of AS II by chloroketone . Alkylation of AS II by iodoacetamide is unaffected by AS I and chorismate . These results suggest a role of chorismate-AS I complex to promote binding of glutamine to AS II or to facilitate conversion of an AS II-glutamine complex to the covalent glutamyl-AS II intermediate . This conclusion is supported by the fact that glutaminase activity of AS II, which requires formation of the glutamyl-AS II intermediate, is stimulated by AS I and chorismate. J Gen Microbiol, 1976 Feb, 92(2), 296 - 303 Intracellular peptide hydrolysis by Pseudomonas putida and Pseudomonas maltophilia; Cascieri T et al.; Amino acids liberated by peptidase hydrolysis of di- and oligopeptides by Pseudomonas putida were measured by trinitrobenzenesulphonate assay and high voltage electrophoresis or paper chromatography followed by ninhydrin spray . Intact bacteria or periplasmic contents released by lysozyme treatment did not hydrolyse peptides . Subcellular fractionation showed that glycylmethionine peptidase activity was cytoplasmic . This enzyme had a Km of 2 mM, and was stimulated fivefold by I mM-Co2+ . Crude peptidase extract did not cleave peptides with D-residues, acylated N-terminal amino groups or N-methylated peptide bonds but otherwise showed a wide specificity . Di- or tripeptides with blocked C-terminus were hydrolysed . Leucylleucine (12 mM) and leucylglycylglycine (10 mM) did not compete with glycylmethionine (1-2 mM) and glycylmethionylglycine (1-0 mM), respectively, for hydrolysis . Pseudomonas maltophilia also contained peptidase activity (0-84 mumol amino acid released from glycylmethionylglycine/min/mg protein) . Peptidases of both P . putida and P . maltophilia were constitutive. J Gen Microbiol, 1976 Feb, 92(2), 283 - 95 Peptide utilization by Pseudomonas putida and Pseudomonas maltophilia; Cascieri T et al.; Pseudomonas putida assimilates peptides and hydrolyses them with intracellular peptidases . Amino acid auxotrophs (his, trp, thr or met) grew on a variety of di- and tripeptides up to twice as slowly as with free amino acids . Pseudomonas putida has separate uptake systems for both dipeptides and oligopeptides (three or more residues) . Although the dipeptide system transported a variety of structurally diverse dipeptides it did not transport peptides having either unprotonatable N-terminal amino groups, blocked C-terminal carboxyl groups, D-residues, three or more residues, N-methylated peptide bonds, or beta-amino acids . Oligopeptide uptake lacked amino acid side-chain specificity, required a free N-terminal L-residue and had an upper size limit . Glycylglycyl-D,L-p-fluorophenylalanine inhibited growth of P . putida . Uptake of glycylglycyl{I-14C}alanine was rapid and inhibited by 2,4-dinitrophenol . Both dipeptide and oligopeptide uptake were constitutive . Dipeptides competed with oligopeptides for oligopeptide uptake, but oligopeptides did not compete in the dipeptide system . Final bacterial yields were 5 to 10 times greater when P . putida his was grown on histidyl di- or tripeptides rather than on free histidine because the histidyl residue was protected from catabolism by L-histidine ammonia-lyase . Methionine peptides could satisfy the methionine requirements of P . maltophilia . Generation times on glycylmethionine and glycylmethionylglycine were equal to those obtained with free methionine . Methionylglycylmethionylmethionine gave a generation time twice that of free methionine . Growth of P . maltophilia was inhibited by glycylglycyl-D,L-p-fluorophenylalanine. J Bacteriol, 1976 Feb, 125(2), 475 - 88 Properties of an inducible uptake system for beta-ketoadipate in Pseudomonas putida; Ornston LN et al.; Wild-type strains of Pseudomonas putida form an inducible uptake system that appears to act on beta-ketoadipate under normal physiological conditions . The system is induced by beta-ketoadipate and is represented by catabolites derived from it . Adipate is metabolized very slowly by wild-type P . putida cultures; {14C}adipate was used as an analogue of beta-ketoadipate to measure the transport activity in wild-type cells and in cells that constitutively produced the uptake system . Constitutive cells that contained high levels of the uptake system concentrated adipate to a level up to 200-fold above the concentration in the external medium . The process was energy dependent . The activity of the system with radioactive adipate was inhibited by beta-ketoadipate, by beta-ketoadipate analogues, and by some compounds (e.g., acetate, glucose) that are structurally unrelated to beta-ketoadipate; it is not known if the inhibitory effects are exerted directly by the compounds themselves or indirectly by catabolites derived from the compounds . The discovery of the beta-ketoadipate uptake system is surprising in view of earlier studies that had indicated that beta-ketoadipate does not permeate the membrane of wild-type P . putida cells . Contradictions between the former investigations and the present analysis are due primarily to the relatively high concentrations of substrate used in the earlier experiments . The existence of the beta-ketoadipate uptake system indicates that beta-ketoadipate may exist as a selective nutrient in the natural niche of P . putida and may play a determinative role in the evolution of induction mechanisms that are characteristic of fluorescent pseudomonads. J Bacteriol, 1976 Feb, 125(2), 404 - 8 Role and regulation of the ortho and meta pathways of catechol metabolism in pseudomonads metabolizing naphthalene and salicylate; Barnsley EA; The enzymes of naphthalene metabolism are induced in Pseudomonas putida ATCC 17484, PpG7, NCIB 9816, and PG and in Pseudomonas sp . ATCC 17483 during growth on naphthalene or salicylate; 2-aminobenzoate is a gratuitous inducer of these enzymes . The meta-pathway enzymes of catechol metabolism are induced in ATCC 17483 and PPG7 during growth on naphthalene or salicylate or during growth in the presence of 2-aminobenzoate, but in ATCC 17484 and NCIB 9816 the ortho-pathway enzymes of catechol metabolism are induced during growth on naphthalene or salicylate . 2-Aminobenzoate does not induce any enzymes of catechol metabolism in the latter two organisms . In Pseudomonas PG the meta-pathway enzymes are present at high levels under all conditions of growth, but this organism and PpG7 can induce ortho-pathway enzymes during naphthalene or salicylate metabolism . Salicylate appears to be the inducer of the enzymes of naphthalene metabolism in all of the organisms studied and, where they are inducible, of the meta-pathway enzymes, but the properties of Pseudomonas PG suggest that separate, regulatory systems may exist. J Gen Microbiol, 1976 Feb, 92(2), 375 - 83 Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida; Condon S et al.; Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment . The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase) . No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P . putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P . putida A90 . Aspartate carbamoyltransferase in crude extracts of P . putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate. Biochim Biophys Acta, 1976 Jan 20, 420(1), 8 - 26 Cytochrome P450cam and its complexes . Mössbauer parameters of the heme iron; Sharrock M et al.; Mossbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida . Native, camphor-free P450cam contains low-spin ferric iron, part of which (approx . 50-70%) is converted to the high-spin ferric state upon addition of camphor . The Mossbauer spectra of the camphor-free enzyme (S equals 1/2) and of the high-spin component (S equals 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple convalency considerations . The native low-spin ferric state of P450cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mossbauer spectra . These changes can be accounted for consistently in the crystal-field model referred to above . The addition of putidaredoxin to the camphor-complexed, oxidized P450cam decreases the intensity of the high-spin component and changes its quadrupole splitting . The reduced form of P450cam contrins high-spin ferrous iron, both in the presence and absence of camphor . The complex of reduced P450cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin . These results are compatible with the bound superoxide, Fe3+-O-2, model proposed for oxyhemoglobin (Weiss, J . J . (1964) Nature 202, 83-84) . Reduced P450cam and its complexes, oxyP450cam-CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes. Eur J Biochem, 1976 Jan 2, 61(1), 259 - 69 Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC; Ohta Y et al.; The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity . Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates . Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric . The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished . Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors . Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared . The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth. Biochim Biophys Acta, 1975 Dec 4, 414(2), 133 - 45 A kinetic and structural characterization of adenosine-5'-triphosphate: ribonucleic acid adenylyltransferase from Pseudomonas putida; Blakesley RW et al.; A catalytic and structural study of ATP:RNA adenylyltransferase (EC 2.7.7.19) from the particulate fraction of Pseudomonas putida was made . During the large-scale purification of this enzyme, designated adenylyltransferase B, a previously undetected ATP-incorporating activity, designated adenylyltransferase A, was observed . Adenylyltransferases A and B were indistinguishable catalytically; however, they differed in their chromatographic and sedimentation properties . Adenylyltransferases A and B were resolved by phosphocellulose, by poly (U)-Sepharose and by Bio-Gel P-100 chromatographies . Adenylytransferase A was determined to have a sedimentation coefficient (S020,w) of 9.3 S and B of 4.3 S . The molecular weight of adenylyltransferase A was estimated to be 185000 and that of adenylyltransferase B to be 50000-60000 . Apparently, adenylyltransferase A was generated from adenylyltransferase B during the purification . The AMP incorporation catalyzed by adenylyltransferases A and B was inhibited by two derivatives of the antibiotic rifamycin, AF/013 (50% at 5 mug/ml) and AF/DNFI (50% at 10 mug/ml) . The 5'-triphosphate derivative (3'-dATP) of the drug cordycepin (3'-deoxyadenosine/ was a competitive inhibitor with ATP for both adenylyltransferases . The Ki for 3'-deoxyadenosine 5'-triphosphate was 6 - 10(-4)--10 - 10(-4) M, while the Km for ATP was 1 - 10(-4)--2 - 10(-4) M . Several other anaolgs of ATP, 2'-deoxyadenosine 5' triphosphate, 2'-O-methyl ATP, or the fluorescent 3-beta-D-ribofuranosylimidazo {2,1-i} purien 5'-triphosphate did not affect the activity of adenylyltransferase A or B . Poly(U) and poly(dT) were competitive inhibitors of the ribosomal RNA-primed polymerization reaction . The Ki for poly(U) or poly(dT), in terms of nucleotide phosphate, was 4 - 10-6)--10 - 10(-6) M for adenylyltransferases A and B, compared to 2 - 10(-4)--4 - 10(-4) M for the Km of ribosomal RNA . The inhibition was a result of the competition between the non-priming poly(U), or poly(dT), and ribosomal RNA for the primer binding site on the enzyme. J Bacteriol, 1975 Dec, 124(3), 1374 - 81 Alternative routes of aromatic catabolism in Pseudomonas acidovorans and Pseudomonas putida: gallic acid as a substrate and inhibitor of dioxygenases; Sparnins VL et al.; When 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) was added to Pseudomonase acidovorans growing at the expense of succinate, enzymes required for degrading homoprotocatechuate to pyruvate and succinate semialdehyde were strongly induced . These enzymes were effectively absent from cell extracts of the organism grown with 4-hydroxyphenylacetic acid, and this substrate was metabolized by the catabolic enzymes of the homogentisate pathway . Two separate ring-fission dioxygenases for 3,4,5-trihydroxybenzoic acid (gallic acid) were present in cell extracts of Pseudomonas putida when grown with syringic acid, and gallate was degraded by reactions associated with meta fission . One of the two gallate dioxygenases also attacked 3-O-methylgallic acid; the other, which did not, was induced when cells were exposed to gallate . This organism possessed ortho fission enzymes, including protocatechuate 3,4-dioxygenase (EC 1.13.11.3) and cis,cis-carboxymuconate-lactonizing enzyme (EC 5.5.1.2), after induction with 3,4-dihydroxybenzoic acid (protocatechuic acid) . Gallate was a substrate for protocatechuate 3,4-dioxygenase, with a Vmax about 3% of that of protocatechuate and with an apparent Km slightly lower . Gallate was a powerful competitive inhibitor of protocatechuate oxidation. J Bacteriol, 1975 Dec, 124(3), 1302 - 11 Physiological consequences of starvation in Pseudomonas putida: degradation of intracellular protein and loss of activity of the inducible enzymes of L-arginine catabolism; Fan CL et al.; We investigated the degradation of radioisotopically labeled intracellular protein in starved, intact cells of Pseudomonas putida P2 (ATCC 25571) and the regulation of this process . Intracellular protein isotopically labeled with L-{4,5-3H}leucine during log-phase growth at 30 C is degraded at rates of 1 to 2%/h in log-phase cells and 7 to 9%/h in starved cells . Rifampin, chloramphenicol, and tosyllysine chloromethylketone lower the rate of protein degradation by starved cells . Addition to starved cells of a nutrient upon which the culture is induced for growth rapidly lowers the rate of protein degradation from 7 to 9%/h to less than 1.5%/h . A nutrient that is oxidized but that cannot immediately support growth also lowers the rate of starvation-induced protein degradation . Proteolytic activity of cell extracts requires a divalent metal ion and may be inhibited up to 60% by tosyllysine chloromethylketone or p-toluenesulfonyl fluoride . Rifampin and chloramphenicol have no effect . In contrast to intact cells, extracts of growing or starving cells degrade protein at equivalent rates . We also investigated the stabilities of the inducible transport system and of four inducible intracellular enzymes of L-arginine catabolism . These include: the membrane-associated, L-arginine-specific transport system; L-arginine oxidase (oxidase); alpha-ketoarginine decarboxylase (decarboxylase); gamma-guanidinobutyraldehyde dehydrogenase ( dehydrogenase); and gamma-guanidinobutyrate amidinohydrolase (hydrolase) . In starved cells, the rates of loss of activities were: transport and dehydrogenase activities, stable; oxidase and decarboxylase activities, 20 to 30%/h; hydrolase activity, 5 to 8%/h . Chloramphenicol decreases the rate of loss of oxidase, decarboxylase, and hydrolase activity, whereas p-toluenesulfonyl fluoride lowers the rate of loss of decarboxylase but not of oxidase or hydrolase activity . Addition to starved cells of a nutrient for which they are already induced for growth (e.g., malate, a noninducer of arginine catabolic enzymes) decreases the rate of loss of oxidase and decarboxylase activity but not that of the hydrolase. Appl Microbiol, 1975 Dec, 30(6), 1046 - 7 Improved method of selection for mutants of Pseudomonas putida; Carhart G et al.; Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved D-cycloserine selection. J Biochem (Tokyo), 1975 Dec, 78(6), 1321 - 9 D-alpha-Hydroxyglutarate dehydrogenase of Rhodospirillum rubrum; Ebisuno T et al.; D-alpha-Hydroxyglutarate dehydrogenase of R . rubrum grown anaerobically in the light was partially purified and some properties were investigated . 1 . The enzyme catalyze stoichiometrically the dehydrogenation reaction of D-alpha-hydroxyglutarate into alpha-oxoglutarate, coupled with the reduction of 2, 6-dichlorophenolindophenol . 2 . Cytochrome c2, cytochrome c, and ferricyanide are effective as electron acceptors with the crude enzyme but not with the purified one, whereas NAD+ and NADP+ are completely ineffective . The enzyme is thought to play a role in the electron transport system of the organism . 3 . D-alpha-Hydroxyglutarate is virtually the sole substrate for the enzyme . The apparent activity against L-alpha-hydroxyglutarate is presumed to be due to contamination of the L-isomer sample with the D-isomer . The enzyme shows barely detectable activity against both isomers of malate and virtually no activity against DL-lactate and glycolate . 4 . Both isomers of malate and oxalate, which are presumably substrate analogues, inhibit the enzyme activity . 5 . The enzyme is not an inducible enzyme but rather is a constitutive one for R . rubrum, unlike from the enzyme of Pseudomonas putida which is an inducible enzyme for the catabolism of lysine. Mol Gen Genet, 1975 Nov 24, 141(2), 113 - 9 Host factor for coliphage Qbeta RNA replication is present in Pseudomonas putida; DuBow MS et al.; Host Factor (HF)1, is a 12000 molecular weight polypeptide that is found in uninfected Escherichia coli and is required as a hexamer along with Qbeta replicase for in vitro replication of Qbeta phage RNA . It has recently been found to be associated with ribosomes and to bind tightly to poly(A) . We report here the identification and purification of HF from Pseudomonas putida . HF can be detected in crude extracts by both functional activity in the Qbeta RNA replication assay and by immunodiffusion with antibody made against E . coli HF . HF from E . coli and P . putida chromatograph similarly on DEAE-cellulose and phosphocellulose . They have similar but not identical molecular weights as judged by SES-polyacrylamide gel electrophoresis . Like E . coli HF, P . putida HF was found to be associated with ribosomes and to bind tightly to poly(A) . Furthermore, the pure protein from P . putida has full funcitonal activity in the in vitro Qbeta RNA replication assay . The findings that HF has been conserved during evolution, is associated with ribosomes, and binds poly(A), suggest that HF may be an important translational element in uninfected cells and that its role involves an interaction with RNA. J Biochem (Tokyo), 1975 Nov, 78(5), 1105 - 7 Exchange reactions catalyzed by methioninase from Pseudomonas putida; Ito S et al.; Highly purified methioninase from Pseudomonas putida, which catalyzes alpha, gamma-elimination reactions of homocysteine and its S-substituted derivatives as well as alpha, beta-elimination reactions of cysteine and its derivatives, was found to catalyze exchange reactions between the substituent at the gamma-carbon of homocysteine substrates and exogenously added alkanethiols, forming the corresponding S-alkylhomocysteines . It also catalyzed similar beta-exchange reactions between cysteine and alkanethiols . Thus, all the substrates for the methioninase-catalyzed elimination reactions also appear to be available for the exchange reactions. Biotechnol Bioeng, 1975 Oct, 17(10), 1485 - 1514 New mechanisms for the biosynthesis and metabolism of 2-keto-L-gulonic acid in bacteria; Makover S et al.; L-Sorbose is oxidized to 2-keto-L-gulonic acid (KGA) via the following sequence of reactions which we call the "sorbosone pathway": L-sorbose in equilibrium L-sorbosone leads to KGA . The first step is reversible and is mediated by enzymes found in a soluble fraction obtained from Pseudomonas putida ATCC 21812 . Although no cofactor requirements were found for the forward reaction, the reverse reaction clearly required NADH . Enzymes for this NADH-dependent synthesis of L-sorbose could be differentiated on the basis of molecular weights . The second step in the sorbosone pathway is catalyzed by a particulate enzyme found in extracts from P . putida and Gluconobacter melanogenus IFO 3293 . The rate limiting reaction in the sorbosone pathway is the synthesis of L-sorbosone . In addition to P . putida, Klebsiella pneumoniae (ATCC 27858) and Serratia marcescens (ATCC 27857) also contain the enzymes which catalyze the reactions of the sorbosone pathway . Two of the bacteria studied, P . putida and G . melanogenus, also contain an enzyme involved in the further metabolism of KGA to L-idonic acid . This enzyme, referred to as KGA-reductase, is found in the soluble fraction of cell-free extracts and is dependent on NADH or NADPH.
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