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| J Bacteriol, 1975 Oct, 124(1), 7 - 13 Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid; Worsey MJ et al.; Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL . These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid . The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene . Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes . They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene. Biochemistry, 1975 Sep 23, 14(19), 4159 - 66 Mössbauer investigations of high-spin ferrous heme proteins . II . Chloroperoxidase, horseradish peroxidase, and hemoglobin; Champion PM et al.; Reduced samples of chloroperoxidase, horseradish peroxidase, and deoxyhemoglobin were studied by Mossbauer spectroscopy in strong magnetic fields . The intricate paramagnetic spectra of chloroperoxidase were evaluated in detail in the framework of a spin Hamiltonian pertinent to high-spin ferrous iron . The studies strongly suggest that, in their reduced states, chloroperoxidase from Caldariomyces fumago and cytochrome P-450 from Pseudomonas putida have similar, if not identical ligand structures of the heme iron . The spectral similarities of these two proteins, noted in an earlier Mossbauer investigation, are further explored and substantiated . Reduced horseradish peroxidase and deoxyhemoglobin, on the other hand, show high-field Mossbauer spectra that differ considerably from each other and, in particular, from those of the P-450 type, suggesting a different ligand arrangement of the heme iron for each case. Biochemistry, 1975 Sep 23, 14(19), 4151 - 8 Mössbauer investigations of high-spin ferrous heme proteins . I . Cytochrome P-450; Champion PM et al.; Anaerobically reduced samples of cytochrome P-450 from Pseudomonas putida were studied by Mossbauer spectroscopy . In the presence of an applied magnetic field the high-spin ferrous heme iron showed an intricate pattern of electric and magnetic hyperfine interactions which could be parametrized successfully in terms of a spin Hamiltonian formalism . The results imply a very low (triclinic) symmetry of the heme iron . The effects of the ligand environment and of spin-orbit coupling result in a large zero-field splitting of the electronic ground state . The electronic ground state . The electric-field gradient tensor is characterized by a large asymmetry parameter, and its principal axes are rotated substantially from the frame that defines the zero-field splitting . This study shows that high-field Mossbauer spectroscopy provides a unique tool for structural investigations of high-spin ferrous compounds and can substitute, under suitable conditions, for magnetic susceptibility measurements . The present paper focuses on the methodology and data analysis; in the subsequent paper the data obtained for P-450 are compared with new results obtained for hemoglobin, chloroperoxidase, and horseradish peroxidase. J Biol Chem, 1975 Sep 10, 250(17), 6870 - 4 The stereochemistry at carbon 3 of pyruvate lyase condensation products . 2-Keto-3-deoxygluconate-6-phosphate aldolase; Meloche HP et al.; In a condensation between {3-3H3}pyruvate and D-glyceraldehyde-3-P as catalyzed by 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) of Pseudomonas putida, C--C synthesis occurred appreciably faster than C--3H bond breaking . Since tritium is present in tritiated pyruvate in tracer amounts, this result showed hydrogen isotope discrimination in pyruvate deprotonation and suggests enolpyruvate generation to be at least partially rate-limiting in the condensation reaction . Consequently, in a condensation reaction between {3-3H, 2H,H}pyruvate of known chirality and D-glyceraldehyde-3-P, the newly synthesized C--C bond would be enriched for at what was the C--H bond of chiral pyruvate, discriminating against the C--2H and C--3H bonds . Additional studies showed that condensations between (3S)-{3-3H, 2H,H}- or (3R)-{3-3H, 2H,H}pyruvate and D-glyceraldehyde-3-P yielded predominantly (3S)- or (3R)-2-keto-3-deoxy{3-3H, 2H}gluconate-6-P, respectively . By comparison with sterochemical models, it was concluded that condensation occurred with retention of configuration at C-3 . Thus in the turnover of substrates as catalyzed by this enzyme, both the exchanging proton from water and D-glyceraldehyde-3-P attack the same face of the enzyme-bound pyruvyleneamine. Biochem J, 1975 Sep, 149(3), 553 - 7 The stereospecificity of sequential nicotinamide-adenine dinucleotide-dependent oxidoreductases in relation to the evolution of metabolic sequences; do Nascimento KH et al.; The generalization that 'when a metabolic sequence involves consecutive nicotinamide-adenine dinucleotide-dependent reactions, the dehydrogenases have the same stereospecificity' was tested and confirmed for three metabolic sequences . (1) NAD+-xylitol (D-xylulose) dehydrogenase and NADP+-xylitol (L-xylulose) dehydrogenase are both B-specific . (2) D-Mannitol 1-phosphate dehydrogenase and D-sorbitol 6-phosphate dehydrogenase are both B-specific . (3) meso Tartrate dehydrogenase and oxaloglycollate reductive decarboxylase are both A-specific . Other dehydrogenases associated with the metabolism of meso-tartrate in Pseudomonas putida, such as hydroxypyruvate reductase and tartronate semialdehyde reductase, were also shown to be A-specific . Malate dehydrogenase from Pseudomonas putida was A-specific, and the proposition is discussed that the common A-stereospecificity among the dehydrogenases involved in meso-tartrate metabolism reflects their origin from malate dehydrogenase. J Bacteriol, 1975 Sep, 123(3), 1088 - 106 Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen; Woolfolk CA; Pseudomonas putida, strain 40, originally isolated by enrichment on caffeine as the sole source of carbon and nitrogen, has been developed to grow on 0.5% caffeine . The organism will grow on any N-methyl derivative of xanthine containing one or more methyl groups at the 1, 3, or 7 positions . An investigation of the activities of resting cell suspensions and cell-free preparations together with the detection of metabolic intermediates suggest that caffeine is first metabolized by the action of an enzyme which is capable of hydrolytically removing all three methyl groups with the production of methanol and free xanthine . The methanol presumably is oxidized to the final product, CO2, through the sequential action of methanol, formaldehyde, and formate dehydrogenases, which are induced by growth on caffeine . Furthermore, the xanthine would seem to be channeled through conventional pathways of purine degradation through the action of xanthine dehydrogenase and uricase, both induced by growth on caffeine . However, a variety of data suggests that the metabolism of caffeine may be compartmentalized in the cell and metabolized separately from externally added xanthine . Additional studies indicated that the cell is permeable to the methylxanthines . The significance of these findings is discussed. Proc Natl Acad Sci U S A, 1975 Sep, 72(9), 3647 - 51 Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA; Chakrabarty AM et al.; Conditions optimal for the transformation of Pseudomonas putida and E . coli with a drug-resistance factor (RP 1) DNA, which specifies resistance to carbenicillin, tetracycline, kanamycin, and neomycin, are described . The transformants retain all the fertility, incompatibility, and drug-resistance characteristics present in the parent . Covalently-closed circular molecules of almost identical contour lengths have been isolated from the parent and the transformants . The frequency of transformation is drastically reduced by treatment of RP 1 DNA with DNase and by denaturation or sonication . Shearing of RP 1 DNA in vitro and their subsequent introduction in P . putida cells, by transformation, produces transformants that exhibit a wide range of drug-resistant phenotypes, including those which are resistant to neomycin but sensitive to kanamycin . Isolation of such neomycin-resistant but kanamycin-sensitive transformants indicates that there might be two separate mechanisms specified by RP 1 for resistance to the two antibiotics. Eur J Biochem, 1975 Sep 1, 57(1), 241 - 56 A 4-methoxybenzoate O-demethylase from Pseudomonas putida . A new type of monooxygenase system; Bernhardt FH et al.; A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-demethylation of this substrate . The enzyme system is purifiable and can be separated into two components: an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase . The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0 . Increasing ionic strength affects these activities differently . The absolute spectrum of the oxidized displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm . Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm . Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores . The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90 . This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical . The optical absorption properties are somewhat unusual . In contrast to other iron-sulfur proteins, there is no significant peak near 415 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm . The presence of the substrate 4-methoxybenzoate increases both the NADH-dependent reductase . Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants . This enzyme opens a new group of oxygenases within the classification scheme, i.e., iron-containing and labile-sulfur-containing monooxygenases . From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed. J Biol Chem, 1975 Aug 25, 250(16), 6445 - 51 Pseudomonas putida cytochrome P-450 . The effect of complexes of the ferric hemeprotein on the relaxation of solvent water protons; Griffin BW et al.; With pulsed nuclear magnetic resonance techniques, the effects of various complexes of ferric cytochrome P-450 on the relaxation rate of bulk solution water protons have been determined . For the camphor, metyrapone, and 4-phenylimidazole complexes, the experimental results are consistent with outer sphere relaxation effects . However, for the substrate-free enzyme, the magnitude and temperature dependence of the paramagnetic relaxation effects indicate the presence of exchangeable protons in the coordination sphere of the heme iron atom . The exchange rate (9.3 x 10(4) S-1 at 25 degrees) and the thermodynamic activation parameters for the exchange process are very similar to those of acid metmyoglobin and acid methemoglobin, suggesting that a water molecule, and not an amino acid residue of the protein, coordinates to the ferric cation of the enzyme in the absence of added substrate or ligands . From the equations appropriate for coordination sphere protons, the distance between these protons and the ferric heme cation was evaluated as 2.1 A, which further supports the interpretation . These experimental results demonstrate that the solvent accessibility of the ferric cation of substrate-free cytochrome P-450 is significantly reduced by the binding of substrate or nitrogenous ligands to the hemeprotein. Biotechnol Bioeng, 1975 Aug, 17(8), 1211 - 35 Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures; Yang RD et al.; The microbial degradation of phenol by pure and mixed cultures of Pseudomonas putida was studied in batch, phenol-stat, and continuous culture systems . In the continuous culture runs, both steady state and transient experiments were performed . From these experiments, a model for the kinetic behavior of the organisms was evolved and an analysis performed on the stability and dynamic behavior of pure and mixed cultures . The results indicate that it should be possible to achieve phenol removal from wastewaters down to levels of 1-2 ppm in a single state system . However, because of the effect of substrate inhibition on kinetic behavior of the microorganisms, long lasting transients can occur . The transient behavior of such systems cannot be solely determined from mumax or Ks parameters, but must include a consideration of the transient size and response characteristic of the organism. J Bacteriol, 1975 Aug, 123(2), 759 - 60 Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonas putida; Benson S et al.; In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase proteins in mutants lacking the ability to convert n-alkanes to their primary alcohols . Purified heptane is an effective inducer in a mutant lacking detectable hydroxylase activity. J Bacteriol, 1975 Aug, 123(2), 546 - 56 Regulation of alkane oxidation in Pseudomonas putida; Grund A et al.; We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid . Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids . Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P . putida chromosome . Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain . Some inducers are neither growth nor respiration substrates . Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity . Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants . This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities. J Biol Chem, 1975 Jul 25, 250(14), 5322 - 9 Intermediates and enzymes between alpha-ketoarginine and gamma-guanidinobutyrate in the L-arginine catabolic pathway of Pseudomonas putida; Vanderbilt AS et al.; In Pseudomonas putida P2 grown on L-arginine as the sole source of carbon and nitrogen, catabolism of L-arginine forms of alpha-ketoarginine, gamma-guanidinobutyrate, and gamma-aminobutyrate . A previously undetected intermediate, gamma-guanidinobutyraldehyde, is identified as the product of alpha-ketoarginine decarboxylase . An 86-fold purification of this enzyme is described . Activity is thiamine pyrophosphate-dependent and cofactor reassociation is facilitated by divalent cations . The order of effectiveness is Mn-2+ greater than Mg-2+, Co-2+ greater than Ca-2+ greater than Ni-2+ greater than Zn-2+ . An inducible enzyme that catalyzes conversion of gamma-guanidinobutyraldehyde to gamma-guanidinobutyrate has been studied in cell-free extracts . NAD-+, but no other cofactors, is required . By differential nutritional growth experiments, 4 regulatory units for the L-arginine pathway are proposed and inducers of 2 units are identified. J Biol Chem, 1975 Jun 25, 250(12), 4580 - 3 Crystallization and properties of L-arginine deiminase of Pseudomonas putida; Shibatani T et al.; Crystalline L-arginine deiminase of Pseudomonas putida was prepared by the following steps: sonic disruption, ammonium sulfate fractionation, protamine sulfate treatment, DEAE-cellulose column chromatography, and L-arginine-Sepharose 6B chromatography followed by crystallization . This procedure yields a crystalline pure enzyme with a 45% recovery of the activity in crude cell-free extracts . The yield is significantly higher than that reported for this enzyme . The purified enzyme appears to be homogeneous in ultracentrifugation (s-o20, w equals 10.2 S) and isoelectric focusing (pI equals 6.13) . The purified enzyme showed two bands on disc gel electrophoresis, both carrying out the deimination of L-arginine . Electrophoresis in the presence of beta-mercaptoethanol plus Na dodecyl-SO4 gave a single band (Mr, 54,000) . Specific activity of this enzyme was 58.8 mumol of L-citrulline formed per min per mg of protein at 37 degrees . The optimum pH of the purified enzyme was 6.0 and maximal activity was obtained at 50 degrees . The molecular weight of the native protein was 130,000 by gel filtration and 120,000 by sedimentation-equilibrium measurements . The spectrum of the pure enzyme showed absorption maximum at 280 nm and the value of E-1%-1 CM AT 280 NM WAS 10.48 IN 0.05 M potassium phosphate buffer (pH 7.0) . The crystalline enzyme hydrolyzed several L-arginine analogues . L-Homoarginine, L-alpha-amino-gamma-guanidinobutyric acid, and L-alpha-amino-beta-guanidinopropionic acid competitively inhibited the hydrolysis of L-arginine with Ki values of 25.7, 7.5, and 4.0 times 10- minus 3 M, respectively . p-Chloromercuribenzoate, Ag-+, and Hg-2+, and several metal ions inhibited the enzyme. J Biol Chem, 1975 May 25, 250(10), 3814 - 25 Metabolism of resorcinylic compounds by bacteria . Purification and properties of orcinol hydroxylase from Pseudomonas putida 01; Ohta Y et al.; Orcinol hydroxylase (EC 1.14.13.6), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized . Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone . The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm . FAD can be dissociated from the protein . Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN . The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein . K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY . The molecular activity of the crystalline enzyme is 1560 min minus 1 . In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2 . Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol . Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated . The latter compounds are not hydroxylated . Instead H2O2 accumulates as the only product of O2 reduction . The enzyme therefore behaves either as a hydroxylase or an oxidase . The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation . The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol . K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy . The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol . The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol . Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector . Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur . Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector . The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation. J Biol Chem, 1975 May 25, 250(10), 3826 - 30 Metabolism of resorcinylic compounds by bacteria . Purification and properties of acetylpyruvate hydrolase from Pseudomonas putida 01; Davey JF et al.; Acetylpyruvate hydrolase, the terminal inducible enzyme of the pathway of orcinol catabolism in Pseudomonas putida, catalyzes the quantitative conversion of acetylpyruvate into acetate and pyruvate . The enzyme has been purified approximately 40-fold from extracts of Ps . putida grown on orcinol . Disc gel electrophoresis of the preparations show one major and one minor band of protein . The molecular weight of the enzyme is approximately 38,000 by sodium dodecyl sulfate electrophoresis . Acetylpyruvate is the only known substrate for the enzyme; maleylpyruvate, fumarylpyruvate, acetoacetate, oxalacetate, and acetylacetone are not hydrolyzed by acetylpyruvate hydrolase . Several divalent cations, includ-Mg2+, Mn2+, Co2+, Ca2+, and Zn2+, enhanced hydrolytic activity, but Cu2+ was inhibitory . The enzyme shows a sharp pH optimum at 7.4 . Acetylpyruvate hydrolase has an apparent K-m of 0.1 mM for acetylpyruvate with a molecular activity of 36 min minus 1 at 25 degrees . Pyruvate, oxalacetate, and oxalate are competitive inhibitors of acetylpyruvate hydrolysis by the enzyme with K-i values of 6.0, 4.5, and 0.45 mM, respectively. Mol Cell Biochem, 1975 Apr 30, 7(1), 59 - 64 The uptake of glucose and gluconate by Pseudomonas putida; Vicente M et al.; The uptake of glucose and gluconate is under inductive control in Pseudomonas putida . Glucose, gluconate, and 2-ketogluconate were each good nutritional inducers of these transport abilities . Glucose and gluconate uptake obeyed saturation kinetics: the apparent Km for glucose was 6 mM and that for gluconate was 0.5 mM . Therefore, transport of both substrates appears to be mediated by enzyme-like carriers . Glucose and gluconate are parallel inhibitors for their uptake9 Strains selected for their inability totransport glucose were found to be deficient in gluconate uptake . The reverse was alsotrue: mutations affecting gluconate entry also blocked the uptake of glucose . These results demonstrate that a common carrier is involved in the uptake of both glucose and gluconate by P . putida cells. J Bacteriol, 1975 Apr, 122(1), 1 - 6 Pathways for the degradation of m-cresol and p-cresol by Pseudomonas putida; Hopper DJ et al.; A comparison of the oxidation rates of various compounds by whole cells of Pseudomonas putida 3, 5 indicated that m-cresol is metabolized by oxidation to 3-hydroxybenzoate followed by hydroxylation to gentisate, the ring-fission substrate, when grown with 3, 5-xylenol . However, when m-cresol was the growth substrate, similar experiments suggested a different pathway involving a methyl-substituted catechol, and ring-fission by meta cleavage . Assays of ring-fission enzymes in cell-free extracts confirmed that different pathways are induced by the two growth substrates . 3, 5-Xylenol-grown cells contained high levels of gentisate oxygenase and only very small amounts of catechol oxygenase, whereas gentisate ocygenase could not be detected in m-cresol-grown cells, but levels of catechol oxygenase were greatly increased . Extracts of m-cresol-grown cells also contained 2-hydroxymuconic semialdehyde dehydrogenase and hydrolase, whose specificities enable them to metabolize the ring-fission products from catechol, 3-methylcatechol, and 4-methylcatechol . This catechol pathway is also used by m-cresol-grown cells for p-cresol metabolism . In contrast, the results for cells grown with p-cresol point to an alternative pathway involving oxidation to 4-hydroxybenzoate and hydrosylation to protocatechuate as ring-fission substrate . Extracts of these cells contained high levels of protocatechuate oxygenase and only small amounts of catechol oxygenase. Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1249 - 53 Autogenous regulation of the inducible tryptophan synthase of Pseudomonas putida; Proctor AR et al.; Mutants blocked before indole-3-glycerol phosphate formation in the tryptophan biosynthetic pathway of P . putida ("early-blocked" mutants) are unable to use indole as a source of tryptophan for growth on minimal medium . The uninduced level of tryptophan synthase {EC 4.2.1.20; L-serine hydro-lyase (adding indole)} in such mutants was thought to be responsible for this property . We have shown that levels of indole higher than those previously tested will support growth of these mutants . In addition, the growth rate of these mutants on a given indole concentration was shown to be proportional to the synthase level induced under the same conditions . This apparent induction of tryptophan synthase by indole in "early-blocked" mutants was shown to be caused by formation of the normal effector molecule, indole-3-glycerol-P, from indole . Secondary mutations occur in "early-blocked" trp strains, which enable them to grow on low concentrations of indole . One type of "indole-utilization" mutation occurs in the trpA gene, inactivating its product . Tryptophan synthase is readily induced by low concentrations of indole in these mutants, even though they are unable to convert indole to indole-3-glycerol-P . We propose that the alpha-chain of the synthase has an autogenous regulatory function, serving as the repressor or the indole-3-glycerol-P recognition component of the repressor of the trpAB operon (synthase alpha-and beta-chains) . Our hypothesis holds that the trpA type of "indole-utilization" mutation alters the repressor (synthase alpha-chain) so that indole as well as indole-3-glycerol-P serves as an effector molecule for tryptophan synthase induction. J Bacteriol, 1975 Apr, 122(1), 93 - 8 Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids; Nieder M et al.; Pseudomonas putida PpG6 is able to utilize purified n-alkanes of six to ten carbon atoms for growth . It can also grow on the primary terminal oxidation products of these alkanes and on 1-dodecanol but not on the corresponding 2-ketones or 1,6-hexanediol, adipic acid, or pimelic acid . Revertible point mutants can be isolated which have simultaneously lost the ability to grow on all five n-alkane growth substrates but which can still grow on octanol or nonanol . An acetate-negative mutant defective in isocitrate lysase activity is unable to grow on even-numbered alkanes and fatty acids . Analysis of double mutants defective in acetate and propionate or in acetate and glutarate metabolism shows that alkane carbon is assimilated only via acetyl-coenzyme A and propionyl-coenzyme A . These results support the following conclusions: (i) The n-alkane growth specificity of P . putida PpG6 is due to the substrate specificity of whole-cell alkane hydroxylation; (ii) there is a single alkane hydroxylase enzyme complex; (iii) the physiological role of this complex is to initiate the monoterminal oxidation of alkane chains; and (iv) straight-chain fatty acids from butyric through nonanoic are degraded exclusively by beta-oxidation from the carboxyl end of the molecule. Biochim Biophys Acta, 1975 Mar 28, 386(1), 87 - 98 Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b-5 and cytochrome P-450-cam; Vickery L et al.; Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats . The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b-5 and P-450, which dominate the spectra . The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome -b-5 from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450-cam from Pseudomonas putida . The magnetic circular dichroism spectra of the membrane bound cytochrome -b-5 are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure . The soluble bacterial cytochrome P-450 also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes . No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed. Biochemistry, 1975 Mar 25, 14(6), 1131 - 9 Mandelate racemase from Pseudomonas putida . Magnetic resonance and kinetic studies of the mechanism of catalysis; Maggio ET et al.; The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated . The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant . Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM . Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz . From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1 . The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+ . Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand . The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments . In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes . From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-{13C}-D,L-mandelate and 2-{13C}-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex . In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array . The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate . This intervening water ligand may polarize or protonate the carboxyl group . From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis . Relationships among the microscopic rate constants are considered. J Biol Chem, 1975 Mar 10, 250(5), 1723 - 33 Purification and characterization of bacteriophage gh-I-induced deoxyribonucleic acid-dependent ribonucleic acid polymerase from Pseudomonas putida; Towle HC et al.; Infection of Pseudomonas putida by the bacteriophage gh-L-induced the synthesis of a novel DNA-dependent RNA polymerase . This gh-L-induced RNA polymerase was purified to near homogeneity . It was shown to be distinct from the host RNA polymerase (alpha-2 beta beta sigma) physically and in respect to many of its catalytic properties . The gh-L-induced RNA polymerase was composed of a single polypeptide of approximately 98,000 molecular weight . The divalent metal ion requirement for in vitro RNA synthesis by the gh-L-polymerase could be satisified with Mg-2+, but not with Mn-2+ . Rna synthesis by the gh-L polymerase was highly resistant to inhibition by rifampicin and streptolydigin but could be inhibited by relatively low concentrations of KCl or the rifamycin derivative AF/013 . The structural analog of ATP, 3'-deoxyadenosine 5'-triphosphate, inhibited both the gh-L-induced and the host RNA polymerases by competing for a single binding site with ATP . The phage polymerase was extremely sensitive to this inhibitor, exhibiting an apparent K-i value (2 times 10-8 M) approximately 100 times lower than that for the host RNA polymerase . The gh-L polymerase had a highly specific template requirement for DNA from the homologous gh-L phage . It would not efficiently utilize denatured DNA templates and had only low levels of activity with pyrimidine-containing polydeoxyribonucleotide homopolymers. Arch Microbiol, 1975 Mar 10, 102(3), 275 - 9 Physiological role for the membrane bound ascorbate-TMPD oxidase in pseudomonas putida; Jones MV; The activity of the membrane-bound ascorbate-TMPD oxidase in Pseudomonas putida varies with growth conditions and age of the culture . A comparison of the effects of cyanide and azide on the oxidation of various substrates suggests that ascorbate-TMPD oxidase is not the terminal oxidase for NADH or succinate oxidation . However, it does have a role in the oxidation of nicotinate, and may act as an additional terminal oxidase under certain other growth conditions. Biochim Biophys Acta, 1975 Feb 19, 377(2), 444 - 53 Catalytic and thermodynamic properties of the urocanate hydratase reaction; Cohn MS et al.; Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor . In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined . As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible . DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively . Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C . Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C . Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C. Biochemistry, 1975 Feb 11, 14(3), 575 - 84 Initial reactions in the oxidation of naphthalene by Pseudomonas putida; Jeffrey AM et al.; A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil . A mutant strain of this organism, P . putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium . The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry . Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P . Fluorescens (formerly ATCC, 17483), P . putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P . putida described in this report . Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor . A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate . Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P . putida . The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase . This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium. J Gen Microbiol, 1975 Feb, 86(2), 217 - 27 Characterization of the growth of Pseudomonas putida LP on lipoate and its analogues: transport, oxidation, sulphur source, and enzyme induction; Shih JC et al.; Pseudomonas putida LP, which grows on lipoate, NH4NO3 and mineral salts, converts most of the organic substrate to bisnor-lipoate (1,2-dithiolane-3-propanoic acid) and acetyl-CoA . D-, L-, or DL-lipoate serve equally well as carbon and sulphur sources . There was no growth on or bacterial oxidation of the chemically synthesized bisnor- or tetranor-(1,2-dithiolane-3-carboxylic acid) chain-shortened analogues, but these, as well as lipoate, could supply the sulphur needed for growth when acetate was provided as the sole source of carbon . The uptake of lipoate by the bacterium is very slow and non-inducible, while the uptake of acetate is faster than octanoate . The oxidation of octanoate is more rapid and extensive than that of lipoate . Levels of acyl-CoA synthetase are not affected by the source of carbon, but activities of isocitrate lyase and malate synthase are higher when the cells are grown in acetate, octanoate or lipoate and lower when glucose is the carbon source . The glyoxylate cycle is induced to facilitate utilization of acetyl-CoA derived from lipoate, which is also degraded to water-soluble catabolites that yield the much smaller amount of sulphur required for growth. Adv Exp Med Biol, 1975, 58(00), 47 - 53 Immunochemical and compositional comparison of cytochrome P-450 cam of Pseudomonas putida and P-450 lm of phenobarbital-induced rabbit liver microsomes; Dus K et al.; Although highly purified cytochrome P-450 of Pseudomonas putida (P-450 cam), and that from phenobarbital-induced rabbit liver microsomes (P-450 LM), differ markedly in their catalytic and physical properties, they show immunological cross reaction by competitive binding and inhibition of catalytic activity, and are of similar amino acid composition . Upon treatment with cyanogen bromide they yield small heme-containing peptides of highly similar amino acid composition. Arch Microbiol, 1975, 102(2), 163 - 6 The uptake of fructose by Pseudomonas putida; Vicente M; Fructose transport was not apparently affected in a number of Pseudomonas putida strains with deranged activity of a common glucose-gluconate uptake system, indicating the existence of an independent fructose uptake system . Fructose uptake by glucose-gluconate uptake mutants was induced by fructose and obeyed saturation kinetics (apparent Km equal 0.3 mM) . The fructose uptake system serves to transport glucose in addition to fructose . The entry of fructose into P.putida cells appears to be mediated also by the glucose-gluconate uptake system, as shown by the ability to accumulate fructose of wild type cells grown on glucose, a substrate that induces the glucose-gluconate uptake system but not the fructose uptake system . In addition, fructose was found to be an inducer of the glucose-gluconate uptake system . The physiological significance of these observations is not clear because the fructose uptake system can provide the cell with a high enough internal concentration of fructose to support maximum growth rate on this hexose, as shown by following the growth course of a glucose-gluconate uptake mutants on fructose.
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