Catabolism of 5-chlorosalicylate by a Bacillus isolated from the Mississippi River.

A strain of Bacillus brevis isolated from a polluted section of the Mississippi River was shown to utilize 5-chloro-2-hydroxybenzoate (5-chlorosalicylate) as a sale source of carbon and energy. Enzymic analyses of cell-free extracts prepared from 5-chlorosalicylate-grown cells demonstrated that the initial step in the pathway involved cleavage of the aromatic ring between C1 and C2 by a specific 5-chlorosalicylate 1,2-dioxygenase. Loss of chloride from the growth substrate occurred after ring fission and was probably enzyme mediated. An intermediate chlorolactone apparently lost chloride by enzymatic hydrolysis with formation of maleylpyruvate. Maleylpyruvate was further degraded by both glutathione-dependent and glutathione-independent mechanisms, with these reactions being identical to the terminal reactions of the gentisate pathway. It was suggested that this novel 5-chlorosalicylate pathway may have evolved by recruitment of enzymes from an ancestral gentisate pathway.     

Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.     

Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans

In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and (1)H and (13)C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.     

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into ‘upper’ (carbaryl to salicylate), ‘middle’ (salicylate to gentisate) and ‘lower’ (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).     

Anthranilic acid metabolism by microorganisms. Formation of 5-hydroxyanthranilate as an intermediate in anthranilate metabolism byNocardia opaca

5-Hydroxyanthranilic acid was isolated and characterized as an intermediate in the metabolism of anthranilate byNocardia opaca. This compound appeared during the log phase of growth and led to the subsequent induction of high levels of enzymes for its subsequent degradation. Gentisate was also found as a product of 5-hydroxyanthranilate metabolism. Both 5-hydroxyanthranilate and gentisate were oxidized to pyruvate by extracts of anthranilate-grown cells, the ring cleavage of the diphenol being effected by an oxygenase with a typical Fe²⁺ requirement. Features of the metabolism of 5-hydroxyanthranilate and gentisate byN. opaca, which can also oxidize anthranilate through catechol, suggest that the gentisate pathway is a secondary route.     

Gentisate 1,2-dioxygenase from pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans

The 3-hydroxybenzoate inducible gentisate 1,2-dioxygenases have been purified to homogeneity from P. acidovorans and P. testosteroni, the two divergent species of the acidovorans group of Pseudomonas. Both enzymes exhibit a 40-fold higher specific activity than previous preparations and have an (alpha Fe)4 quaternary structure (holoenzyme Mr = 164,000 and 158,000, respectively). The enzymes have different amino terminal sequences, amino acid contents, and isoelectric points. Each enzyme contains essential active site iron that is EPR silent but binds nitric oxide quantitatively to give an EPR active complex (S = 3/2), showing that the iron is Fe2+ with coordination sites for exogenous ligands. The EPR spectra of these complexes are altered uniquely for each enzyme when gentisate is bound. This suggests that substrate binds to or near the iron and shows that the substrate-iron interactions of each enzyme are subtly different. The kinetic parameters for turnover of gentisate by the enzymes are nearly identical (kcat/Km = 4.3 x 10(6) s-1 M-1). Both enzymes cleave a wide range of gentisate analogs substituted in the 3 or 4 ring position, although at reduced rates relative to gentisate. Of the two enzymes, P. testosteroni gentisate 1,2-dioxygenase exhibits substantially lower kcat/Km values for the turnover of these compounds. Evidence for both steric and electronic substituent effects is obtained. In accord with the results of Wheelis et al. (Wheelis, M. L., Palleroni, N. J., and Stanier, R. Y. (1967) Arch. Mikrobiol. 59, 302-314), 3-hydroxybenzoate is shown to be metabolized by P. acidovorans through the gentisate pathway, and gentisate 1,2-dioxygenase is the only ring cleavage dioxygenase induced. In contrast, 3-hydroxybenzoate is metabolized by P. testosteroni exclusively through the protocatechuate pathway utilizing protocatechuate 4,5-dioxygenase, although gentisate 1,2-dioxygenase is coinduced. Growth of P. testosteroni on 3-O-methylbenzoate or 5-O-methylsalicylate is shown to result in a approximately 10-fold increase in the amount of gentisate 1,2-dioxygenase relative to protocatechuate 4,5-dioxygenase. Together, these results suggest that induction of gentisate 1,2-dioxygenase by 3-hydroxybenzoate in P. testosteroni may be adventitious and that this enzyme may function in fundamentally different metabolic pathways in the two related Pseudomonas species.     

Site-directed mutagenesis of gentisate 1,2-dioxygenases from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is the first enzyme in gentisate pathway that catalyses the ring fission of gentisate to form maleylpyruvate. Phylogenetic tree of amino acid sequences from 11 GDOs demonstrates that the GDOs from different genus share identities between 12.1% and 64.8%. According to the alignment result, four highly conserved histidine residues in GDO from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2 were chosen to be substituted with aspartate residues. Enzyme analysis indicated that substitution of any of these four histidine residues had resulted in the complete loss of its catalytic activity.     

Gentisate 1,2-dioxygenase from Pseudomonas. Substrate coordination to active site Fe2+ and mechanism of turnover

Gentisate 1,2-dioxygenase catalyzes the oxygenolytic ring cleavage of gentisate (2,5-dihydroxybenzoate) between carbons 1 and 2 to form maleylpyruvate. The essential active site Fe2+ of the enzyme binds NO to yield an EPR-active (S = 3/2) complex. Hyperfine broadening from 17O (I = 5/2) is observed in the spectrum of the enzyme-nitrosyl complex prepared in 17O-enriched water, demonstrating that water is an iron ligand. Association of gentisate with the enzyme-nitrosyl complex causes the broadening due to [17O]water to disappear, suggesting that water is displaced. Hyperfine broadening of the EPR spectrum for the gentisate-bound complex is observed when 17O is incorporated into either the carbon 1 carboxylate or carbon 2 hydroxyl substituents of gentisate, but not when it is placed in the carbon 5 hydroxyl substituent. Thus, substrate apparently binds directly to the iron through the carbon 1 carboxylate and carbon 2 hydroxyl substituents, thereby bringing the site of ring cleavage close to the active site iron. Since NO must bind to the iron to elicit an EPR signal, a total of three sites in the iron coordination appear to be available for exogenous ligands. The role of the substrate functional groups in catalysis is investigated through comparison of the reaction kinetics of gentisate analogs using the gentisate 1,2-dioxygenases isolated from Pseudomonas acidovorans and Pseudomonas testosteroni. Turnover is either eliminated or substantially reduced on replacement of any of the functional groups of gentisate. Furthermore, an electron-donating group that can tautomerize (hydroxyl or amine) is required in a ring position either ortho or para to the carbon 2 substituent for turnover. The best alternate substrate of this group is 5-aminosalicylate, which is turned over at approximately 7% of the rate of gentisate by the enzyme from P. testosteroni. Both atoms from O2 are shown to be incorporated into the product of 5-aminosalicylate turnover. This is the first direct demonstration of dioxygenase stoichiometry in the reaction of any ferrous, non-heme, aromatic ring-cleaving dioxygenase. It is proposed that the enzyme-catalyzed O2 attack on the aromatic ring of gentisate is initiated from a complex in which O2 and substrate are simultaneously coordinated to the active site iron. Subsequent dioxygen bond cleavage and insertion are proposed to be promoted by a resonance shift involving ketonization of the carbon 5 hydroxyl group.     

Initial reactions of xanthone biodegradation by an Arthrobacter sp.

This study examined the catabolism of xanthone by an Arthrobacter sp. (strain GFB100) capable of growth on xanthone as its main source of carbon and energy. An early catabolic intermediate was 3,4-dihydroxyxanthone. This compound was isolated from the growth medium of a mutant strain of the Arthrobacter sp. which lacked the xanthone-inducible dihydroxyxanthone ring-fission dioxygenase of the wild-type strain. Cell extracts from wild-type xanthone-grown cells oxidized 3,4-dihydroxyxanthone to a yellow ring-fission metabolite. The same yellow compound accumulated in xanthone-grown cultures of a spontaneous mutant which lacked an active, xanthone-inducible, NADPH-linked ring-fission metabolite reductase. The yellow ring-fission metabolite appears to be 4-hydroxy-3-(2'-oxo-3-trans-butenoate)-coumarin, based on its nuclear magnetic resonance spectrum and mass spectral fragmentation pattern, indicating that ring cleavage of 3,4-dihydroxyxanthone was by an extra-diol (meta-fission) mechanism. Enzymatic analyses indicated that growth on xanthone induced a complete gentisate pathway: dioxygenase-catalyzed cleavage of gentisate to maleylpyruvate, isomerization of maleylpyruvate to fumarylpyruvate, and hydrolysis of fumarylpyruvate to fumarate and pyruvate. 4-Hydroxycoumarin was thought to be a likely pathway intermediate linking the early xanthone catabolic steps to the gentisate pathway, since 2-hydroxyacetophenone, a byproduct of 4-hydroxycoumarin hydrolysis, was formed when wild-type cells were cultured with xanthone. Chlorinated 2-hydroxyacetophenones were also obtained from specific chloro-substituted xanthones.     

芳香烃龙胆酸降解途径蛋白质组学的研究

芳香烃是一类重要的环境污染物,微生物降解是其主要的处理方法。研究显示降解过程中产生保守型和诱导型的各一组同工酶。目前,仅有保守型的龙胆酸加双氧酶（GDOI）及其下游片段被克隆。产碱假单胞菌NCIB9867（P25X）的突变株-SNZ28 GDOI被打断,在龙胆酸诱导的情况下,该突变株仍能检测到龙胆酸加双氧酶活性。采用二维蛋白电泳分析突变株SNZ28在有和没有龙胆酸诱导条件下的蛋白质表达差异。电泳结果显示了两者存在有15个蛋白点的差异。通过MALDI-TOF和Q—TOF分析,其中的12个蛋白质点与数据库中已知多肽片段有同源性。其中,P4点与青枯菌（Ralstonia species）龙胆酸1,2加双氧酶同源。该结果在蛋白质组学上证实了GDOII的存在。     

Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867

Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.     

Salicylate 5-hydroxylase from Ralstonia sp. strain U2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase

The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the "classical" naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.     

Metabolism of carbaryl via 1,2-dihydroxynaphthalene by soil isolates Pseudomonas sp. strains C4, C5, and C6

Pseudomonas sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O2 uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl --> 1-naphthol --> 1,2-dihydroxynaphthalene --> salicylaldehyde --> salicylate --> gentisate --> maleylpyruvate.     

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.     

Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Deltancg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacterium Cupriavidus necator JMP134

Cupriavidus necator JMP134 is a model for chloroaromatics biodegradation, capable of mineralizing 2,4-D, halobenzoates, chlorophenols and nitrophenols, among other aromatic compounds. We performed the metabolic reconstruction of aromatics degradation, linking the catabolic abilities predicted in silico from the complete genome sequence with the range of compounds that support growth of this bacterium. Of the 140 aromatic compounds tested, 60 serve as a sole carbon and energy source for this strain, strongly correlating with those catabolic abilities predicted from genomic data. Almost all the main ring-cleavage pathways for aromatic compounds are found in C. necator : the β-ketoadipate pathway, with its catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage branches; the (methyl)catechol meta ring-cleavage pathway; the gentisate pathway; the homogentisate pathway; the 2,3-dihydroxyphenylpropionate pathway; the (chloro)hydroxyquinol pathway; the (amino)hydroquinone pathway; the phenylacetyl-CoA pathway; the 2-aminobenzoyl-CoA pathway; the benzoyl-CoA pathway and the 3-hydroxyanthranilate pathway. A broad spectrum of peripheral reactions channel substituted aromatics into these ring cleavage pathways. Gene redundancy seems to play a significant role in the catabolic potential of this bacterium. The literature on the biochemistry and genetics of aromatic compounds degradation is reviewed based on the genomic data. The findings on aromatic compounds biodegradation in C. necator reviewed here can easily be extrapolated to other environmentally relevant bacteria, whose genomes also possess a significant proportion of catabolic genes.     

Drastic changes in the peptidoglycan composition of penicillin resistant laboratory mutants of Streptococcus pneumoniae

Abstract Rhodococcus erythropolis strain S1 uses the gentisate pathway to metabolize salicylate and m -hydroxybenzoate and the protocatechuate pathway to degrade p -hydroxybenzoate. m -Hydroxybenzoate 6-hydroxylase was induced by growth on m -hydroxybenzoate or gentisate, and salicylate 5-hydroxylase only by growth on salicylate. p -Hydroxybenzoate 3-hydroxylase could be induced only by growth on p -hydroxybenzoate. m -Hydroxybenzoate or p -hydroxybenzoate could repress the induction of salicylate 5-hydroxylase. Maleylpyruvate isomerase in the gentisate pathway did not require reduced glutathione.     

Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869

Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1, 2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1, 2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Kms of 92 and 143 microM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with kcat/Km values of 44.08 x 10(4) s-1 M-1 for the P25X enzyme and 39.34 x 10(4) s-1 M-1 for the P35X enzyme. Higher kcat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1, 2-dioxygenases was around 50 degrees C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.     

Metabolism of Carbaryl via 1,2-Dihydroxynaphthalene by Soil Isolates Pseudomonas sp. Strains C4, C5, and C6

Pseudomonas sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O₂ uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl → 1-naphthol → 1,2-dihydroxynaphthalene → salicylaldehyde → salicylate → gentisate → maleylpyruvate.