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Showing papers on "Pseudomonas putida published in 1980"


Journal ArticleDOI
TL;DR: All strains of Pseudomonas aeruginosa produced a distinctive series of odd-carbon methyl ketones, particularly 2-nonanone and 2-undecanone, and2-aminoacetophenone, which were present in strains of P. aerug inosa and in variable amounts in other species.
Abstract: Gas chromatographic-mass spectrometric analysis of headspace volatiles was performed on cultures of 11 strains of Pseudomonas aeruginosa and 1 strain each of Pseudomonas cepacia, Pseudomonas putida, Pseudomonas putrefaciens, Pseudomonas fluorescens, and Pseudomonas maltophilia. All strains of Pseudomonas aeruginosa produced a distinctive series of odd-carbon methyl ketones, particularly 2-nonanone and 2-undecanone, and 2-aminoacetophenone. The other strains failed to produce 2-aminoacetophenone. Two sulfur compounds, dimethyldisulfide and dimethyltrisulfide, were present in strains of P. aeruginosa and in variable amounts in other species. Butanol, 2-butanone, 1-undecene, and isopentanol were also detected in P. aeruginosa cultures.

157 citations


Journal ArticleDOI
TL;DR: Derivatives of Pseudomonas sp.
Abstract: Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.

132 citations


Journal ArticleDOI
TL;DR: Results showed that two methoxyl groups of 3,4,5-trimethoxybenzoate and one of syringate were oxidized to give carbon dioxide and 3-O-methylgallate, which was presumed to be the 4-methyl ester of oxalacetic acid, for which cell extracts contained an inducible, specific esterase.
Abstract: When grown at the expense of 3,4,5-trimethoxybenzoic acid, a strain of Pseudomonas putida oxidized this compound and also 3,5-dimethoxy-4-hydroxybenzoic (syringic) and 3,4-dihydroxy-5-methoxybenzoic (3-O-methylgallic) acids; but other hydroxy- or methoxy-benzoic acids were oxidized slowly or not at all. Radioactivity appeared exclusively in carbon dioxide when cells were incubated with [4-methoxyl-14C]trimethoxybenzoic acid, but was found mainly in methanol when[methoxyl-14C]3-O-methylgallic acid was metabolized. The identity of methanol was proved by analyzing the product from [methoxyl-13C]3-O-methylgallic acid by nuclear magnetic resonance spectroscopy and by isolating the labeled 3,5-dinitrobenzoic acid methyl ester, which was examined by mass spectrometry. These results, together with measurements of oxygen consumed in demethylations catalyzed by cell extracts, showed that two methoxyl groups of 3,4,5-trimethoxybenzoate and one of syringate were oxidized to give carbon dioxide and 3-O-methylgallate. This was then metabolized to pyruvate; the other product was presumed to be the 4-methyl ester of oxalacetic acid, for which cell extracts contained an inducible, specific esterase. P. putida did not metabolize the methanol released from this compound by hydrolysis. Support for the proposed reaction sequence was obtained by isolating mutants which, although able to convert 3,4,5-trimethoxybenzoic acid into 3-O-methylgallic acid, were unable to use either compound for growth.

58 citations


Journal ArticleDOI
TL;DR: The results demonstrate that mutants may be selected showing altered enzyme activities in the absence of acquiring the capacity to grow solely on a novel substrate which provided the selection pressure.
Abstract: A continuous-flow culture selection experiment was designed to obtain mutants of Pseudomonas putida strain PP3 capable of growing on 2-monochlorobutanoic acid (2MCBA), normally unable to act as a carbon and energy source for P putida PP3 The experiment involved continuous exposure of P putida PP3 to 2MCBA whilst the population's growth was supported by the metabolizable substrate, 2-monochloropropionic acid The system failed to select for mutants able to grow on 2MCBA alone However, the procedure resulted in the selection of two different strains of P putida, namely, PP309 and PP310, both stably maintained as a mixed culture having excluded the parent strain The two mutants differed significantly from P putida PP3 with respect to their dehalogenating system since it was found that major changes in both dehalogenase specific activities and overall activity ratios had occurred The dehalogenase system in P putida strains PP309 and PP310 was inducible as in the parent strain The results obtained were explained in terms of elevated production of fraction I dehalogenase compared with fraction II dehalogenase in the mutant strains in contrast to the parent strain The results demonstrate that mutants may be selected showing altered enzyme activities in the absence of acquiring the capacity to grow solely on a novel substrate which provided the selection pressure

57 citations


Journal ArticleDOI
TL;DR: It is proposed that enzymes of the 3,5-xylenol pathway and those for conversion of p-cresol to p-hydroxybenzoate are plasmid encoded, that the early methyl-oxidizing enzymes are expressed constitutively, and that the later enzymes are inducible.
Abstract: Constitutive synthesis of enzymes responsible for methyl group oxidation in 3,5-xylenol degradation and an associated p-cresol methylhydroxylase in Pseudomonas putida NCIB 9869 was shown by their retention at high specific activities in cells transferred from 3,5-xylenol medium to glutamate medium. The specific activities of other enzymes of the 3,5-xylenol pathway declined upon removal of aromatic substrate, consistent with their inducible control. Specific activities of the methyl-oxidizing enzymes showed an eventual decline concomitant with a decrease in the fraction of bacteria capable of growth with 3,5-xylenol; a simultaneous loss of the ability to grow with m-hydroxybenzoate was also observed. The property of 3,5-xylenol utilization could be transferred to another strain of P. putida. It is proposed that enzymes of the 3,5-xylenol pathway and those for conversion of p-cresol to p-hydroxybenzoate are plasmid encoded, that the early methyl-oxidizing enzymes are expressed constitutively, and that the later enzymes are inducible. Images

48 citations


Journal ArticleDOI
TL;DR: 1,2-Dihydroxynaphthalene oxygenase was purified from Pseudomonas putida NCIB 9816, leading to the appearance of 2-hydroxychromene-2-carboxylic acid, but 3-methylcatechol was oxidized by this enzyme to 2-Hydroxy-6-oxoheptadienoic acid, which may indicate that 2- Hydroxy chromene- 2-car boxylic Acid results from
Abstract: 1,2-Dihydroxynaphthalene oxygenase was purified from Pseudomonas putida NCIB 9816 grown on naphthalene as the sole source of carbon and energy. The enzyme had a subunit molecular weight of 19,000 and in a medium containing phosphate buffer, 1 mM mercaptoethanol, and 10% (vol/vol) ethanol had a native molecular weight greater than 275,000. The enzyme required Fe2+ for activity. It was inactivated slowly on standing, and inactivation was accelerated by dilution with aerated buffers and by H2O2. Bathophenanthroline sulfonate, o-phenanthroline, 8-hydroxyquinoline, and 2,2'-dipyridyl also inhibited the enzyme. The inactive enzyme was reactivated by anaerobic incubation with ferrous sulfate and ferrous ammonium sulfate. Thiol reagents and acetone, ethanol, or glycerol decreased the rate of loss of activity. The enzyme was most active with 1,2-dihydroxynaphthalene, for which the Km was 2.8 X 10(-4) M. 3-Methyl- and 4-methylcatechols were oxidized at 3 and 1.5%, respectively, of the rate of 1,2-dihydroxynaphthalene, and the Km for 3-methylcatechol was 1.5 X 10(-4) M. Purified 1,2-dihydroxynaphthalene oxygenase catalyzed the oxidation of 1,2-dihydroxynaphthalene, leading to the appearance of 2-hydroxychromene-2-carboxylic acid, but 3-methylcatechol was oxidized by this enzyme to 2-hydroxy-6-oxoheptadienoic acid. Thus, a product structurally analogous to 2-hydroxychromene-2-carboxylic acid was not observed when 3-methylcatechol was oxidized. This may indicate that 2-hydroxychromene-2-carboxylic acid results from cyclization of a ring fission product before release from the enzyme.

45 citations



Journal ArticleDOI
TL;DR: Two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria are developed and high yields of GS are obtained.
Abstract: We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.

39 citations


Journal ArticleDOI
TL;DR: The amino acid sequence of anthranilate synthase component II (AS II) from Serratia marcescens was determined and the active site region is virtually identical to that of the Pseudomonas putida AS II enzyme.

37 citations


Journal ArticleDOI
TL;DR: The formaldehyde generated by the demethylation of caffeine is oxidized by an NAD-dependent formaldehyde dehydrogenase, which is independent of Mg2+ and glutathione, which means that the enzyme is absolutely dependent on NADH or NADPH as a cosubstrate and activated by CO2+.
Abstract: 1) The enzymatic demethylation of caffeine (1,3,7-trimethylxanthine) by Pseudomonas putida C1 was investigated; an inducible enzyme system has been observed. This enzyme shows an optimum pH of about 6.0, and the optimum temperature is in the range of 22-24 degrees C. The enzyme is absolutely dependent on NADH or NADPH as a cosubstrate and is activated by CO2+. 2) The formaldehyde generated by the demethylation of caffeine is oxidized by an NAD-dependent formaldehyde dehydrogenase, which is independent of Mg2+ and glutathione. The enzyme was purified from cell-free extracts of Pseudomonas putida C1 by DEAE-cellulose, Sephadex G-150 and Sephadex A-50 chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and was most active at a pH between 8.5 and 9.0. The molecular weight was estimated to be about 250,000 by the gel filtration method. Kinetic analysis gave KM values of about 0.2 mM for formaldehyde and 0.5 mM for NAD+.

34 citations


Journal ArticleDOI
TL;DR: It is suggested that the defective mutants are enriched because of the genetic alterations they possess, which confer immunity to a lethal synthesis performed by transformation of the analogs in clones possessing an intact p-cymene pathway.
Abstract: Several classes of mutants of Pseudomonas putida (JT810) defective in the utilization of p-cymene as sole carbon source have been isolated. Selective enrichment of the mutants and for strains putatively cured of a degradative plasmid was achieved by incubation of cells in minimal growth media containing p-cymene (or p-cumate) and various halogenated analogs of the growth substrates or pathway intermediates. Analogs which led to successful enrichments included: p-chlorotoluene, p-bromotoluene, alpha-chloro-p-xylene, and p-iodobenzoate. A mutant strain, PpJT811, constitutive for the p-cymene pathway gave significantly greater enrichments of defective mutants than the wild-type parent PpJT810 after incubation with the halogenated analogs. It is suggested that the defective mutants are enriched because of the genetic alterations they possess, which confer immunity to a lethal synthesis performed by transformation of the analogs in clones possessing an intact p-cymene pathway. A nomenclature for the genetic organization of p-cymene pathway is described.

Journal ArticleDOI
TL;DR: The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and PseUDomonas putida; in the first two species comparison could be made with Flac.
Abstract: The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.

Journal ArticleDOI
TL;DR: High-resolution pyrolysis gas-liquid chromatography was applied to three bacteria grown under a variety of conditions and showed that four peaks could be used to discriminate the three bacteria however they were cultured.
Abstract: High-resolution pyrolysis gas-liquid chromatography was applied to three bacteria (Escherichia coli NCTC 9001, Pseudomonas putida (NCIB 9494, and Staphylococcus aureus NCTC 8532) grown under a variety of conditions. Changing the culture medium drastically altered the quantitative aspects of the pyrograms of all three organisms, but the effects of culture time and incubation temperature were less severe. Mathematical analysis of the relative peak heights showed that four peaks could be used to discriminate the three bacteria however they were cultured.

Journal ArticleDOI
TL;DR: Two media used for demonstrating alginolytic activity are described and the applied aspects of the ability of these two species to digest algin are discussed.
Abstract: Pseudomonas maltophilia and Pseudomonas putida were identified as alginolytic species. Two media used for demonstrating alginolytic activity are described. The applied aspects of the ability of these two species to digest algin are discussed.

Journal ArticleDOI
TL;DR: The purification of Pseudomonas beta-ketoadipate enol-lactone hydrolase is described and evidence is presented indicating that the protein is a trimer composed of identical 11,000-dalton subunits that favors the interpretation that copies of DNA were substituted into structural genes for the enzymes as they diverged.

Journal ArticleDOI
TL;DR: Over the past several years, researchers have sought to exploit the inherent photochemical reactivity of the A4-3-one competitive inhibitors of the enzyme in an effort to identify, by photochemical modification techniques, important functional groups of the steroid binding active sites of this enzyme.
Abstract: In 1955, Paul Talalay reported the occurrence and partial purification of a AS-3-ketosteroid isomerase (EC 5.3.3.1) in Pseudomonas testosteroni grown in the presence of testosterone.’ By 1959, the isomerase was available in essentially pure crystalline preparations.’ The reaction catalyzed by the isomerase has been shown to consist of an allylic isomerization of steroidal 5-ene-3-ones to their conjugated 4-ene-3-one isomers with concomitant 4p to 68 intramolecular proton transfer. This reaction is shown in FIGURE 1. The enzyme has been the object of numerous investigations of its structure and function. Much of this work has been the subject of a recent review.’ AS-3-Ketosteroid isomerase has attracted interest because of its rather large turnover number, its moderate size, and the fact that it is obtainable in a pure state in large quantities. In addition, because it binds steroids as substrates or competitive inhibitors, albeit with less affinity than mammalian intracellular steroid receptors, some effort has been made to elucidate the structural bases for steroid binding by the isomerase. The primary structure of the constituent polypeptide chain has been reported by Benson et al.4 and a crystallographic study of its threedimensional structure is reported to be ~ n d e r w a y . ~ Below a concentration of approximately 2 mg/ml, the testosteroni isomerase is a dimer of identical 13,394 dalton subunits. Each subunit consists of a single polypeptide chain. The dimer probably has two independent functioning steroid binding although Vincent et a1.* have reported that, in their experiments, the dimer exhibits a “half-of-the-sites’’ binding stoichiometry. Considerable variation of the steroid structure can be tolerated without exceeding the ability of the enzyme to bind the steroid. In a recent comprehensive survey, Weintraub et al.9 found that a wide variety of estrogens, androgens, and progestins were competitive inhibitors of the catalytic process. Many of the enzyme’s competitive inhibitors are steroidal A4-3-ones, the products of the enzymatic reaction when it is conducted in the thermodynamically favored direction. Over the past several years, we have sought to exploit the inherent photochemical reactivity of the A4-3-one competitive inhibitors of the enzyme in an effort to identify, by photochemical modification techniques, important functional groups of the steroid binding active sites of this enzyme.’&’* The most detailed work has been conducted

Journal ArticleDOI
01 May 1980-Plasmid
TL;DR: The transposons Tn 501, Tn 7, and Tn 1 were used to mark 20 FP plasmids of Pseudomonas aeruginosa which have previously been characterized on the basis of their host chromosome mobilizing ability and their contribution to error-prone DNA repair to study the host range, entry exclusion, and incompatibility systems of these plasmid.


Journal ArticleDOI
TL;DR: Incubation of racemic mixtures of dl-(+/-)-nicotine with Pseudomonas putida resulted in a complete stereoselective degradation of the l-(-) isomer, which was not affected by the bacterium and was recovered by extraction.
Abstract: Incubation of racemic mixtures of dl-(+/-)-nicotine with Pseudomonas putida resulted in a complete stereoselective degradation of the l-(-) isomer. Unnatural d-(+)-nicotine, which is of pharmacological interest for stereochemical studies of various nicotine-responsive systems, was not affected by the bacterium and was recovered by extraction.


Journal ArticleDOI
TL;DR: A delta 5-3-ketosteroid isomerase (EC 5.3.1) has been isolated from Pseudomonas putida Biotype B and purified to homogeneity, and the amino acid sequence has been determined for the NH2-terminal 50 residues.

Journal ArticleDOI
TL;DR: De oxygencholate protects the enzyme from the TEO-dependent reaction to a degree which is predictable from the concentrations of deoxycholate and TEO and their respective competitive inhibition constants, thus demonstrating that the inititial velocity of the photoinactivation is dependent upon the fraction of enzyme active sites occupied by TEO.

Journal ArticleDOI
TL;DR: Two new and independently isolated catabolic plasmids coding for the degradation of naphthalene have been characterized in strains of Pseudomonas putida and are transmissible and belong to the P9 incompatibility group.
Abstract: SUMMARY: Two new and independently isolated catabolic plasmids coding for the degradation of naphthalene have been characterized in strains of Pseudomonas putida. Both plasmids are transmissible, belong to the P9 incompatibility group and code for naphthalene degradation via salicylate and catechol, then by the catechol meta-cleavage pathway.

Journal ArticleDOI
D. P. Cox1, A. L. Williams1
TL;DR: In this article, a biological process for converting naphthalene to cis-1,2-dihydroxy-1-2-Dihydronaphthalenes catalyzed by Pseudomonas putida strain 119 was optimized in flask experiments.
Abstract: A biological process for converting naphthalene to cis-1,2-dihydroxy-1,2-dihydronaphthalene (DHD) catalyzed by Pseudomonas putida strain 119 was optimized in flask experiments. These studies revealed the following: (i) P. putida 119 can propagate efficiently and produce DHD when supplied one of several carbon sources and naphthalene; (ii) maximum DHD production by P. putida 119 occurs in logarithmic-growth-phase cells and decreases at various rates in the stationary growth phase, depending upon the carbon source used; (iii) several analogs of salicylic acid can be used as effective inducers of naphthalene metabolism in P. putida cells growing on glucose; and (iv) the addition of chemical surfactants to naphthalene-cell (P. putida 119) mixtures stimulates DHD production.


Journal ArticleDOI
TL;DR: In these mutants, enzymes involved in the conversion of naphthalene to catechol were not produced constitutively but remained inducible during growth on salicylate indicating that these enzymes belong to a separate operon or operons.
Abstract: SUMMARY: The regulation of the catabolic pathway for the degradation of naphthalene specified by the plasmids NAH, pND140 and pND160 was studied using plasmid-borne regulatory mutants in a Pseudomonas putida host strain. Growth of strains harbouring the parent plasmids in the presence of salicylate resulted in induction of selected enzymes involved in the conversion of naphthalene to catechol (naphthalene oxygenase, salicylaldehyde dehydrogenase and salicylate hydroxylase) and enzymes of the meta-cleavage pathway (catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semi-aldehyde dehydrogenase). Partial induction was also observed for all the NAH-encoded enzymes assayed when using m-toluate as the inducing compound, over a genetic block. Mutants were obtained for each plasmid where the three enzymes of the meta-cleavage pathway were produced constitutively suggesting that the enzymes of the meta-cleavage pathway belong to one operon. In these mutants, enzymes involved in the conversion of naphthalene to catechol were not produced constitutively but remained inducible during growth on salicylate indicating that these enzymes belong to a separate operon or operons.

Journal ArticleDOI
TL;DR: The selective value of the β-ketoadipate transport system may lie in its apparent function in chemotaxis and in its ability to control intracellular concentrations of the inducing metabolite,β-ketoadedipate.
Abstract: beta-Ketoadipate induces catabolic enzymes in Pseudomonas putida. The compound is transported by a system which also concentrates adipate, a non-metabolizable analogue of beta-ketoadipate. The natural substrate, beta-ketoadipate, competitively inhibits adipate transport with a K1 of 0.04 mM, lower than the Km of 0.23 mM observed with adipate. Transport is inhibited competitively by succinate (K1 1.3 mM) and non-competitively by acetate (K1 5.3 mM). The system has a sharp pH optimum at 5.5. Transport activity is stimulated by a variety of ions, and salt concentrations in excess of 0.1 M are required to achieve optimal rates of influx. The transport system is inhibited by proton conductors and thiol reagents. Membrane vesicle preparations concentrate adipate when supplied with an oxidizable energy source. Induction of the transport system does not allow the rapid utilization of exogenous beta-ketoadipate. Nevertheless, the system has been conserved in the evolution of divergent Pseudomonas species. The selective value of the beta-ketoadipate transport system may lie in its apparent function in chemotaxis and in its ability to control intracellular concentrations of the inducing metabolite, beta-ketoadipate.

Journal ArticleDOI
TL;DR: The results are consistent with P23X16's being unable to synthesize a functional isomerase while retaining decarboxylase activity and establish the physiological importance of an enzyme-catalyzed isomerization in the meta-cleavage degradation of 4-hydroxyphenylacetate.
Abstract: Degradation of 2-hydroxy-5-carboxymethylmuconic semialdehyde, the ring fission product of the 4-hydroxyphenylacetate meta-cleavage pathway, by mutant strains P23X19 and P23X16 of Pseudomonas putida NCI B 9865 was studied. Both mutants were unable to grow on either 4-hydroxyphenylacetate of 3,4-dihydroxyphenylacetate. Cell extracts of P23X19, grown in the presence of 3,4-dihydroxyphenylacetate, degraded the ring fission product to a compound that accumulated and had maximum UV absorption at 300 nm, pH 7.4, and 345 nm, pH 12. These are the spectral characteristics of 2-keto-5-carboxymethylhex-3-ene-1,6-dioate, the substrate for the decarboxylase in this pathway. This observation is consistent with P23X19's being decarboxylase defective. Cell extracts of P23X16, grown in the presence of 3,4-dihydroxyphenylacetate, degraded the ring fission product to a compound that accumulated and has maximum UV absorption at 295 nm, pH 7.4, and 345 nm, pH 12. This compound spontaneously degraded to a compound with the spectral properties of the decarboxylase substrate. The compound accumulated by P23X16 was also obtained when the decarboxylase substrate was treated with borate. It is suggested that the compound accumulated by P23X16 is the substrate of an isomerase. The results are consistent with P23X16's being unable to synthesize a functional isomerase while retaining decarboxylase activity and establish the physiological importance of an enzyme-catalyzed isomerization in the meta-cleavage degradation of 4-hydroxyphenylacetate.

Journal ArticleDOI
TL;DR: A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct.
Abstract: Incubation of urocanase from Pseudomonas putida with either its substrate, urocanic acid, or product, 4'(5')-imidazolone-5'(4')-propionic acid, resulted in an oxygen-dependent inhibition of enzyme activity. Coincident with the inactivation was the stoichiometric incorporation of radioactivity from [14C]urocanate into the protein. NAD+ which is required for activity or urocanase was not directly involved in the inactivation process. The inactivation of urocanase was irreversible, could be partially blocked by the competitive inhibitor imidazolepropionate, and involved the modification of a single active-site thiol. The inhibition resulted from oxidative decomposition of 4'(5')-imidazolone-5'(4')-propionate but was not due to the formation of the major degradative product, 4-ketoglutaramate, since this compound was not an irreversible inactivator of urocanase although it did produce some inhibition at high concentrations. A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct. These results emphasize the importance of a catalytic center sulfhydryl group for urocanase activity.

Journal ArticleDOI
TL;DR: The reduction stereochemistry of the Schiff's base formed between pyruvate and the ϵ-amino of the catalytic lysine of 2-keto-3-deoxygluconate-6-P-phosphate aldolase of Pseudomonas putida was investigated in this article.