Biochemical and molecular characterization of the gentisate transporter GenK in Corynebacterium glutamicum

Background

Gentisate (2,5-dihydroxybenzoate) is a key ring-cleavage substrate involved in various aromatic compounds degradation. Corynebacterium glutamicum ATCC13032 is capable of growing on gentisate and genK was proposed to encode a transporter involved in this utilization by its disruption in the restriction-deficient mutant RES167. Its biochemical characterization by uptake assay using [¹⁴C]-labeled gentisate has not been previously reported.

Methodology/Principal Findings

In this study, biochemical characterization of GenK by uptake assays with [¹⁴C]-labeled substrates demonstrated that it specifically transported gentisate into the cells with V _max and K_m of 3.06±0.16 nmol/min/mg of dry weight and 10.71±0.11 µM respectively, and no activity was detected for either benzoate or 3-hydoxybenzoate. When GenK was absent in strain RES167 ΔgenK, it retained 85% of its original transport activity at pH 6.5 compared to that of strain RES167. However, it lost 79% and 88% activity at pH 7.5 and 8.0, respectively. A number of competing substrates, including 3-hydroxybenzoate, benzoate, protocatechuate and catechol, significantly inhibited gentisate uptake by more than 40%. Through site-directed mutagenesis, eight amino acid residues of GenK, Asp-54, Asp-57 and Arg-386 in the hydrophobic transmembrane regions and Arg-103, Trp-309, Asp-312, Arg-313 and Ile-317 in the hydrophilic cytoplasmic loops were shown to be important for gentisate transport. When conserved residues Asp-54 and Asp-57 respectively were changed to glutamate, both mutants retained approximately 50% activity and were able to partially complement the ability of strain RES167 ΔgenK to grow on gentisate.

Conclusions/Significance

Our results demonstrate that GenK is an active gentisate transporter in Corynebacterium glutamicum ATCC13032. The GenK-mediated gentisate transport was also shown to be a limiting step for the gentisate utilization by this strain. This enhances our understanding of gentisate transport in the microbial degradation of aromatic compounds.     

mhbR和mhbT基因移码突变对3-羟基苯甲酸代谢的影响

Klebsiella pneumoniae M5a1菌株能以3-羟基苯甲酸作为唯一碳源和能源生长,编码分解代谢3-羟基苯甲酸的基因被克隆至-8kb的DNA片段上(pBSI质粒),含有该质粒的大肠杆菌获得了以3-羟基苯甲酸作为唯一碳源和能源生长的能力。经测序并与GenBank中的已知基因产物进行氨基酸同源性比较后,发现其中的mhbR与转录调节基因和mhbT与底物转运基因具有较高同源性,推断可能具有相应的功能。含有mhbR的移码突变子或mhbT的移码突变子的大肠杆菌在底物3-羟基苯甲酸中培养时,其生长速度和代谢速度都较野生型明显缓慢。     

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.     

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.     

PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida.

PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The Vmax for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the Km was 6 microM. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.     

mhpT Encodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

Escherichia coli K-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome, mhpT was proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated that mhpT is essential for 3HPP catabolism in E. coli K-12 W3110 at pH 8.2. Uptake assays with ¹⁴C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpT containing recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpT containing the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital for E. coli K-12 W3110 growth on this substrate under basic conditions.     

Molecular and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase from Polaromonas naphthalenivorans CJ2

Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI'-orf1-tnpA and nagR2-orf2I'KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.     

Metabolism of 2-, 3- and 4-hydroxybenzoates by soil isolates Alcaligenes sp. strain PPH and Pseudomonas sp. strain PPD

Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.     

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.     

Cloning of the <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. FG1 dehalogenase genes and construction of hybrid pathways in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order.     

Regulation of isofunctional enzymes in Pseudomonas alcaligenes mutants defective in the gentisate pathway

The regulation of the inducible set of gentisate pathway enzymes used by Pseudomonas alcaligenes (P25X1) has been studied in strains derived from mutant strains of P25X1 that had lost the constitutive enzymes that degrade m –cresol, 2,5–xylenol and 3,5–xylenol. The enzyme, 3-hydroxybenzoate 6-hydroxylase II, that catalyzes the oxidation of 3-hydroxybenzoate to gentisate is substrate- and product-induced while gentisate dioxygenase II is substrate induced. Neither 3-hydroxybenzoate nor gentisate could induce the synthesis of maleylpyruvate hydrolase II and fumarylpyruvate hydrolase II. The results suggest that the structural genes encoding these four inducible enzymes and maleylpyruvate hydrolase I (a constitutive enzyme) exist in at least four operons. There is strict induction specificity of expression of this inducible set of gentisate pathway enzymes. 3-Hydroxy-4-methyl-benzoate failed to induce whilst 3-hydroxybenzoate and 3-hydroxy-5-methylbenzoate served as inducers of 6-hydroxylase II. Degradation of 2,5-xylenol is mediated by constitutive enzymes whereas the inducible set of enzymes are responsible for the metabolism of m -cresol and 3,5-xylenol.     

Charged amino acids conserved in the aromatic acid/H+ symporter family of permeases are required for 4-hydroxybenzoate transport by PcaK from Pseudomonas putida

Charged amino acids in the predicted transmembrane portion of PcaK, a permease from Pseudomonas putida that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for P. putida to have a chemotactic response to 4-HBA. An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H+ symporter family of the major facilitator superfamily of transporters.     

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.     

Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867

The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.     

Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation.

A strain of Pseudomonas putida (TMB) was found to resemble P. putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested. The ability of P. putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.     

Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI′-orf1-tnpA and nagR2-orf2I″KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.     

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.     

Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1

3-Hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 is an enzyme that utilizes 3-hydroxybenzoate (3-HBA) as substrate yielding gentisate. Site-directed mutagenesis was carried out to define which residues may be involved in catalytic reaction. Substitution of arginine to glutamate at position 169 of the enzyme resulted in the complete loss of catalytic activity. This indicated Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis.