嗜盐古菌Haloferax volcanii WFD11中龙胆酸1,2-双加氧酶HagA的研究

【目的】研究嗜盐古菌Haloferax volcanii WFD11菌株以不同芳香酸作为碳源的生长情况;鉴定其通过龙胆酸途径代谢芳香酸过程中的开环酶龙胆酸1,2-双加氧酶的基因,并对其进行生化水平的研究;初步揭示古菌和细菌代谢芳香酸的可能差异。【方法】分别以4 mmol/L的6种不同芳香酸为唯一碳源培养菌株WFD11,利用全自动生长曲线分析仪测定菌株生长情况并绘制生长曲线;利用高效液相色谱检测菌株WFD11代谢3-羟基苯甲酸的中间产物;对菌株WFD11的基因组进行生物信息学分析,寻找潜在的龙胆酸1,2-双加氧酶编码基因,并在Haloferax volcanii H1424中异源表达;通过快速纯化系统(采用Ni2+-NTA亲和层析柱)纯化异源表达的蛋白,以龙胆酸为底物通过紫外分光光度计检测粗酶液和纯化后的龙胆酸1,2-双加氧酶和相关酶学特性;通过实时定量PCR观察hag A的表达类型。【结果】菌株WFD11能以4 mmol/L的3-羟基苯甲酸和3-羟基苯丙酸为唯一碳源和能源生长;高效液相色谱检测证明菌株WFD11通过龙胆酸代谢3-羟基苯甲酸(3HBA);克隆和异源表达了龙胆酸1,2-双加氧酶基因hag A;Hag A粗酶液和纯化蛋白均具龙胆酸1,2-双加氧酶的活性,催化龙胆酸开环生成顺丁二酸单酰丙酮酸;Hag A的龙胆酸1,2-双加氧酶比活力为0.024 8 U/mg,且其活性不依赖于Fe2+;荧光定量PCR实验结果证明hag A是组成型表达。【结论】嗜盐古菌H.volcanii WFD11可能是通过龙胆酸途径代谢芳香酸类物质,为进一步研究古菌和细菌代谢芳香酸的可能差异打下了基础。     

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.     

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.     

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.     

nag genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding enzymes for gentisate catabolism

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate to central metabolites. We have cloned and sequenced a 21.6-kb region spanning the nag genes. Upstream of the pathway genes are nagY, homologous to chemotaxis proteins, and nagR, a regulatory gene of the LysR family. Divergently transcribed from nagR are the genes for conversion of naphthalene to gentisate (nagAaGHAbAcAdBFCQED) (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522-2530, 1998), which except for the insertion of nagGH, encoding the salicylate 5-hydroxylase, are homologous to and in the same order as the genes in the classical upper pathway operon described for conversion of naphthalene to salicylate found in the NAH7 plasmid of Pseudomonas putida PpG7. Downstream of nahD is a cluster of genes (nagJIKLMN) which are probably cotranscribed with nagAaGHAbAcAdBFCQED as a single large operon. By cloning into expression vectors and by biochemical assays, three of these genes (nagIKL) have been shown to encode the enzymes involved in the further catabolism of gentisate to fumarate and pyruvate. NagI is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate and is also able to catalyze the oxidation of some substituted gentisates. NagL is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. NagK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The three other genes (nagJMN) have also been cloned and overexpressed, but no biochemical activities have been attributed to them. NagJ is homologous to a glutathione S-transferase, and NagM and NagN are proteins homologous to each other and to other proteins of unknown function. Downstream of the operon is a partial sequence with homology to a transposase.     

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.     

The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum

Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC- and mshD- lost the ability to produce mycothiol, but mutant mshB- produced mycothiol as the wild type did. The phenotypes of mutants mshC- and mshD- were the same as the wild type when grown in LB or BHIS media, but mutants mshC- and mshD- were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 +/- 11.9 microM and 1520 +/- 57.4 micromol/min/mg, respectively (mycothiol concentration, 2.5 microM). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.     

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.     

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.     

Evidence for isofunctional enzymes used in m-cresol and 2,5-xylenol degradation via the gentisate pathway in Pseudomonas alcaligenes.

Study of the reaction sequence by which Pseudomonas alcaligenes (P25X1) and derived mutants degrade m-cresol, 2,5-xylenol, and their catabolites has provided indirect evidence for the existence of two or more isofunctional enzymes at three different steps. Maleylpyruvate hydrolase activity appears to reside in two different proteins with different specificity ranges, one of which (MPH1) is expressed constitutively; the other (MPH11) is strictly inducible. Two gentisate 1,2-dioxygenase activities were found, one of which is constitutively expressed and possesses a broader specificity range than the other, which is inducible. From oxidation studies with intact cells, there appear to be two activities responsible for the 6-hydroxylation of 3-hydroxybenzoate, and again a broadly specific activity is present regardless of growth conditions; the other is inducible by 3-hydroxybenzoate. Three other enzyme activities are also detected in uninduced cells, viz., xylenol methylhydroxylase, benzylalcohol dehydrogenase, and benzaldehyde dehydrogenase. All apparently possess broad specificity. Fumarylpyruvate hydrolase was also detected but only in cells grown with m-cresol, 3-hydroxybenzoate, or gentisate. Mutants, derived either spontaneously or after treatment with mitomycin C, are described, certain of which have lost the ability to grow with m-cresol and 2,5-xylenol and some of which have also lost the ability to form the constitutive xylenol methylhydroxylase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase, 3-hydroxybenzoate 6-hydroxylase, and gentisate 1,2-dioxygenase. Such mutants, however, retain ability to synthesize inducibly a second 3-hydroxybenzoate 6-hydroxylase and gentisate 1,2-dioxygenase, as well as maleylpyruvate hydrolase (MPH11) and fumarylpyruvate hydrolase; MPH1 was still synthesized. These findings suggest the presence of a plasmid for 2,5-xylenol degradation which codes for synthesis of early degradative enzymes. Other enzymes, such as the second 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, maleylpyruvate hydrolase (MPH1 and MPH11), and fumarylpyruvate hydrolase, appear to be chromosomally encoded and, with the exception of MPH1, strictly inducible.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

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 RES167Δncg12918 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.     

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols.

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.     

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.     

Gentisate pathway in Salmonella typhimurium: metabolism of m-hydroxybenzoate and gentisate

Abstract Salmonella typhimurium was shown to use the gentisate pathway to metabolize m -hydroxybenzoate and gentisate. m -Hydroxybenzoate hydroxylase and gentisate 1,2-dioxygenase were induced by growth on either gentisate or m -hydroxybenzoate. These enzymes were not detected when the bacteria were grown with glucose or glucose and either m -hydroxybenzoate or gentisate. However, both enzymes were induced when the bacteria were grown on succinate with either substrate. The maleylpyruvate isomerase required reduced glutathione and was irreversibly inhibited by N -ethylmaleimide.     

Metabolism of 3-hydroxybenzoate and gentisate by strain <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP

The ability of strain Rhodococcus opacus 1CP to utilize 3-hydroxybenzoate (3-HBA) and gentisate in concentrations up to 600 and 700 mg/L, respectively, as sole carbon and energy sources in liquid mineral media was demonstrated. Using high-performance liquid chromatography (HPLC) and thin-layer chromatography, 2,5-dihydroxybenzoate (gentisate) was identified as the key intermediate of 3-hydroxybenzoate transformation. In the cell-free extracts of the strain grown on 3-HBA or gentisate, the activities of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, and maleylpyruvate isomerase were detected. During growth on 3-HBA, low activity of catechol 1,2-dioxygenase was detected. Based on the data obtained, the pathway of 3-HBA metabolism by strain R. opacus 1CP was proposed.     

Novel L-cysteine-dependent maleylpyruvate isomerase in the gentisate pathway of Paenibacillus sp. strain NyZ101

Glutathione- and mycothiol-dependent maleylpyruvate isomerases are known to be involved, respectively, in gentisate catabolism in Gram-negative and high G+C Gram-positive strains. In the present study, a low-G+C Gram-positive Paenibacillus sp. strain, NyZ101, was isolated and shown to degrade 3-hydroxybenzoate via gentisate. A 6.5-kb fragment containing a conserved region of gentisate 1,2-dioxygenase genes was cloned and sequenced, and four genes (bagKLIX) were shown to encode the enzymes involved in the catabolism to central metabolites of 3-hydroxybenzoate via gentisate. The Bag proteins share moderate identities with the reported enzymes in the 3-hydroxybenzoate catabolism, except BagL that had no obvious homology with any functionally characterized proteins. Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration. BagL was demonstrated to be a novel thiol-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate with L-cysteine, cysteinylglycine, or glutathione, as its cofactor. The K(m) values of these three thiols for BagL were 15.5, 8.4, and 552 μM, respectively. Since cysteine and coenzyme A were reported to be abundant in low-G+C Gram-positive strains, BagL should utilize L-cysteine as its physiological cofactor in vivo. The addition of Ni(2+) increased BagL activity, and site-directed mutagenesis experiments indicated that three conserved histidines in BagL were associated with binding to Ni(2+) ion and were necessary for its enzyme activity. BagL is the first characterized L-cysteine-dependent catabolic enzyme in microbial metabolism and is likely a new and distinct member of DinB family, with a four-helix-bundle topology, as deduced by sequence analysis and homology modeling.     

EHEC O157:H7 z3276基因缺失株构建及其生物学特性

【目的】肠出血性大肠杆菌O157:H7是世界范围内重要的动物源性致病菌之一,可感染人。I型菌毛是多种致病性大肠杆菌(如肾盂肾炎型大肠杆菌等)可表达的一种黏附结构,与细菌吸附黏膜表面密切相关。然而,O157:H7 fim操纵子上几个核苷酸的缺失却导致其不能表达I型菌毛。BLAST比对结果表明O157:H7独有的开放阅读框z3276编码的氨基酸序列与其他大肠杆菌I型菌毛高度同源,这可能是对O157:H7不能表达I型菌毛的补偿机制,但确切功能尚不清楚。本文探究z3276基因的生物学功能。【方法】利用O157:H7 86-24参考菌株构建z3276基因缺失株(?z3276),并构建其互补株(C?z3276),进而比较亲本株、?z3276与C?z3276的生物学特性及对小鼠致病性差异。【结果】与亲本株相比,?z3276进入对数生长期的时间延后,在半固体琼脂平板上的迁移直径明显缩小,生物被膜形成能力显著减弱。?z3276对HEp-2细胞的黏附和侵袭能力并无明显变化,但对IPEC-J2细胞的侵袭能力明显减弱。在小鼠攻毒试验中,?z3276组排菌数量减少、排菌持续时间缩短。C?z3276各项特性均能回复到与亲本株一致的水平。【结论】z3276基因可能是O157:H7重要的毒力相关因子。     

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.