Distribution of virulence markers among Vibrio vulnificus isolates of clinical and environmental origin and regional characteristics in Japan.

BACKGROUND: Vibrio vulnificus is an opportunistic human pathogen that is widely distributed in estuarine environments and is capable of causing necrotizing fasciitis and sepsis. In Japan, based on epidemiological research, the incidences of V. vulnificus were concentrated in Kyusyu, mainly in coastal areas of the Ariake Sea. To examine the virulence potential, various genotyping methods have recently been developed. This study aimed to investigate the distribution of virulence markers among V. vulnificus isolates of clinical and environmental origin in three coastal areas with different infection incidences and to determine whether these isolates have the siderophore encoding gene viuB. METHODOLOGY/PRINCIPAL FINDINGS: We examined the distribution of genotypes of the 16S ribosomal ribonucleic acid (rRNA) gene, vvhA, vcg, and capsular polysaccharide (CPS), and the presence of viuB in 156 isolates collected from patients and environmental samples in Japan. The environmental samples were collected from three coastal areas: the Ariake Sea, Ise & Mikawa Bay, and Karatsu Bay. The results showed disparity in the ratios of genotypes depending on the sample origins. V. vulnificus isolates obtained from patients were classified into the clinical type for all genotypes. In the environmental isolates, the ratios of the clinical type for genotypes of the 16S rRNA gene, vvhA, and vcg were in the order of the Ariake Sea>Ise & Mikawa Bay>Karatsu Bay. Meanwhile, CPS analysis showed no significant difference. Most isolates possessed viuB. CONCLUSIONS: Many V. vulnificus belonging to the clinical type existed in the Ariake Sea. Three coastal areas with different infection incidences showed distinct ratios of genotypes. This may indicate that the distribution of clinical isolates correlates with the incidence of V. vulnificus infection.

Crystallization and preliminary X-ray analysis of 4-pyridoxolactonase from Mesorhizobium loti.

4-pyridoxolactonase from Mesorhizobium loti MAFF303099 has been overexpressed in Escherichia coli. The recombinant enzyme was purified and was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 and ammonium sulfate as precipitants. Crystals of the free enzyme (form I) and of the 5-pyridoxolactone-bound enzyme (form II) grew under these conditions. Crystals of form I diffracted to 2.0 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60 A, beta = 117.33 degrees. Crystals of form II diffracted to 1.9 A resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68 A, beta = 118.02 degrees. The calculated V(M) values suggested that the asymmetric unit contains one molecule in both crystal forms.

Gene identification and characterization of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD+-dependent dismutase.

A chromosomal gene, mlr6793, in Mesorhizobium loti was identified as the gene encoding 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) dehydrogenase (dismutase) involved in the degradation pathway for pyridoxine (vitamin B(6)). The homogenously purified recombinant enzyme has a molecular mass of 59.1 kDa and is a homodimeric protein. FHMPC dehydrogenase catalyses practically irreversible oxidation (k(cat) = 204 s(-1)) of FHMPC (K(m) = 48.2 microM) by NAD(+) (K(m) = 34.3 microM) to 3-hydroxy-2-methyl-pyridine 4, 5-dicarboxylic acid (HMPDC), and practically irreversible reduction (k(cat) = 217 s(-1)) of FHMPC (K(m) = 24.9 microM) by NADH (K(m) = 12.4 microM) to 4-pyridoxic acid. When the enzyme reaction was started with the combination of FHMPC and NAD(+) or that of FHMPC and NADH, HMPDC and 4-pyridoxic acid were produced in an almost equimolar ratio throughout the reaction. FHMPC dehydrogenase belongs to the 3-hydroxyacyl-CoA dehydrogenase family with 31% identity with the human enzyme: it has probable catalytic diad residues, i.e. His137 and Glu149. The H137L mutant enzyme showed no measurable activity. The E149Q one was stable in contrast to the corresponding human 3-hydroxyacyl-CoA dehydrogenase mutant, and showed unique pH optima depending on the co-substrates used for the reaction.

Synthesis of 4-pyridoxolactone from pyridoxine using a combination of transformed Escherichia coli cells.

We developed a simple and efficient synthesis for 4-pyridoxolactone starting with pyridoxine and using a whole-cell biotransformation by two transformed Escherichia coli cell types. One set of transformed cells expressed pyridoxine 4-oxidase, catalase, and chaperonin, while the second set expressed pyridoxal 4-dehydrogenase. With this combination of cells, pyridoxine was first oxidized to pyridoxal, which was then dehydrogenated to 4-pyridoxolactone by pyridoxine 4-oxidase and pyridoxal 4-dehydrogenase, respectively. In a reaction mixture containing the two transformed cell types, 10 mM of pyridoxine was completely converted into 4-pyridoxolactone at 30 degrees C in 24 h. When starting with 80 mM of pyridoxine, it was necessary to add 0.5 mM or more of NAD(+) to complete the reaction.

Gene identification and characterization of the pyridoxine degradative enzyme alpha-(N-acetylaminomethylene)succinic acid amidohydrolase from Mesorhizobium loti MAFF303099.

We have found for the first time that a chromosomal gene, mlr6787, in Mesorhizobium loti encodes the pyridoxine degradative enzyme alpha-(N-acetylaminomethylene)succinic acid (AAMS) amidohydrolase. The recombinant enzyme expressed in Escherichia coli cells was homogeneously purified and characterized. The enzyme consisted of two subunits each with a molecular mass of 34,000+/-1,000 Da, and exhibited Km and kcat values of 53.7+/-6 microM and 307.3+/-12 min(-1), respectively. The enzyme required no cofactor or metal ion. The primary structure of AAMS amidohydrolase was elucidated for the first time here. The primary structure of the enzyme protein showed no significant identity to those of known hydrolase proteins and low homology to those of fluoroacetate dehalogenase (PDB code, 1Y37), haloalkane dehalogenase (1K5P), and aryl esterase (1VA4).

Determination of individual vitamin B(6) compounds based on enzymatic conversion to 4-pyridoxolactone.

A determination method for individual natural vitamin B(6) compounds was developed. The vitamin B(6) compounds were specifically converted into 4-pyridoxolactone (PAL), a highly fluorescent compound, through a combination of enzymatic reactions and HCl-hydrolysis. PAL was then determined by HPLC. Pyridoxal was completely oxidized to PAL with pyridoxal 4-dehydrogenase (PLDH). Pyridoxine and pyridoxamine were totally converted into PAL through a coupling reaction involving pyridoxine 4-oxidase and PLDH, and one involving pyridoxamine-pyruvate aminotransferase and PLDH, respectively. The 5'-phosphate forms and pyridoxine-beta-glucoside were hydrolyzed with HCl, and then determined as their free forms. Pyridoxine 5'-phosphate and pyridoxine-beta-glucoside were not separately determined here. Three food samples were analyzed by this method.

Gene identification and characterization of the pyridoxine degradative enzyme 4-pyridoxic acid dehydrogenase from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099.

The gene encoding 4-pyridoxic acid dehydrogenase was identified as mlr6792 in a chromosome of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099. The enzyme is the fourth enzyme in the vitamin B(6) (pyridoxine)-degradation pathway I. The recombinant enzyme with a his-tag over-expressed in Escherichia coli cells was a membrane-bound protein, and purified to homogeneity. The enzyme was a monomeric protein with a molecular weight of 59,000, and a flavoprotein containing one mole of FAD per mole of subunit. The optimum pH and temperature, and K(m) for 4-pyridoxic acid were pH 8.5 and 30 degrees C, and 29 muM, respectively. The enzyme was a glucose-methanol-choline (GMC) family protein with two signature patterns, FAD-binding residues, a putative active site histidine residue and a probable transmembrane segment.

Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099.

Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5'-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P4(3)2(1)2 and was refined to an R factor of 15.6% (R(free) = 20.6%) at 2.0 A resolution. In addition, the structures of PPAT in complexes with pyridoxamine, pyridoxal, and pyridoxyl-L-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (R(free) = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0 A resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The alpha-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5'-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.

Identification of a new tetrameric pyridoxal 4-dehydrogenase as the second enzyme in the degradation pathway for pyridoxine in a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti.

We have found for the first time that a chromosomal gene, mlr6807, in Mesorhizobium loti encodes a new tetrameric pyridoxal 4-dehydrogenase (PLDH). The recombinant enzyme expressed in Escherichia coli cells was homogenously purified and characterized. The enzyme consisted of four subunits each with a molecular weight of 26,000+/-1000, and exhibited Km and kcat values of 91+/-2 microM and 149+/-1s(-1), respectively. PLDH used NAD+ as a cosubstrate, showed no activity toward sugars, and belonged to a short-chain dehydrogenase/reductase family. The mlr6807 gene-disrupted M. loti cells could grow in a nutrient-rich TY medium but not in a synthetic one containing pyridoxine or pyridoxamine as the sole carbon and nitrogen source. Thus, it was found that PLDH is essential for the assimilation of vitamin B6 compounds and the second step enzyme in their degradation pathway in M. loti.

Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase.

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.

Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The gene (mlr6788) of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 has been identified as a gene coding for 2-methyl-3-hydroxypyridine-5-carboxylic acid dioxygenase (MHPCO), the seventh enzyme in degradation pathway I for pyridoxine, a free form of vitamin B(6). The gene was cloned and overexpressed in Escherichia coli cells co-transformed with chaperonin genes. The homogeneous recombinant enzyme showed similar enzymatic properties to the enzyme from Pseudomonas sp. MA-1. MHPCO was essential for the assimilation of pyridoxine in M. loti, but not for its growth in a nutrient-rich medium. From the infection experiment of a symbiotic plant Lotus japonicus with an M. loti mlr6788 gene disruptant, MHPCO was demonstrated to be dispensable for at least nodule formation on roots of seedlings in symbiosis.

4-Pyridoxolactonase from a symbiotic nitrogen-fixing bacterium Mesorhizobium loti: cloning, expression, and characterization.

4-Pyridoxolactonase is involved in the degradation pathway for pyridoxine, a free form of vitamin B6. The gene (mlr6805) encoding the putative 4-pyridoxolactonase of nitrogen fixing symbiotic microorganism Mesorhizobium loti MAFF303099 has been identified based on the genome database. The gene was cloned and overexpressed in a cotransformant Escherichia coli cell. The recombinant enzyme was dimeric protein and contained one mole of Zn2+ per mole of subunit. The enzyme showed about 30% identity with various N-acylhomoserine lactone lactonases and metallo-beta-lactamases. The phylogram made with ClustalW shows that 4-pyridoxolactonase makes a cluster with Agrobacterium tumefaciens acyl-homoserine lactone lactonase. The alignment of amino acid sequences suggests that 4-pyridoxolactonase has three histidine residues probably involved in binding of Zn2+.

Crystallization and preliminary X-ray analysis of pyridoxal 4-dehydrogenase, the second enzyme in degradation pathway I of pyridoxine.

Pyridoxal 4-dehydrogenase (PLDH; EC 1.1.107) is the second enzyme in the bacterial degradation pathway I of vitamin B(6), which catalyzes the oxidation of pyridoxal to 4-pyridoxolactone using NAD(+). PLDH from Microbacterium luteolum, a dimeric protein with a subunit molecular weight of 38 kDa, was crystallized at 277 K in a drop solution comprising 15%(w/v) polyethylene glycol 4000, 0.15 M sodium acetate, 7.5 mM n-octyl-beta-D-glucoside and 0.075 M Tris-HCl pH 7.5 by the sitting-drop vapour-diffusion method. The crystals were monoclinic and belonged to space group C2, with unit-cell parameters a = 107.0, b = 56.7, c = 130.2 A, beta = 103.6 degrees . Diffraction data were collected from a single crystal to 2.0 A.

Molecular cloning, expression, and properties of an unusual aldo-keto reductase family enzyme, pyridoxal 4-dehydrogenase, that catalyzes irreversible oxidation of pyridoxal.

Microbacterium luteolum YK-1 has pyridoxine degradation pathway I. We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase. The gene consists of 1,026-bp nucleotides and encodes 342 amino acids. The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES. The recombinant enzyme showed the same properties as the M. luteolum enzyme. The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR). Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A. tumefaciens d-threo-aldose 1-dehydrogenase and Pseudomonas sp. l-fucose dehydrogenase. These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins. The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars. Furthermore, pyridoxal 4-dehydrogenase prefers NAD(+) to NADP(+) as a cofactor, although AKRs generally show higher affinities for the latter.

The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6.

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a+. Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K(m) values for pyridoxine (213+/-19 microM) and oxygen (78+/-10 microM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.

Coenzyme precursor-assisted cooperative overexpression of an active pyridoxine 4-oxidase from Microbacterium luteolum.

The overexpression system of the active pyridoxine 4-oxidase from Microbacterium luteolum was developed. When chaperonin GroEL/ES genes in plasmid pKY206 were coexpressed, the pyridoxine 4-oxidase gene cloned in the vector pTrc99A was overexpressed in Escherichia coli JM109 cultured in LB medium containing 50microM riboflavin, the precursor of coenzyme (FAD) of the enzyme, under the cold stress at 23 degrees C. The crude extract from the cotransformant cells showed 88-fold higher specific activity than that from M. luteolum. The chaperonins, cold stress, and the riboflavin cooperatively served to increase the soluble form of the enzyme. A significant correlation between the specific activity and percentage of the soluble form in the total expressed enzyme was found. The overexpressed pyridoxine 4-oxidase was easily purified to homogeneity with two steps of the conventional column chromatography.

Inhibition of diphenolase activity of tyrosinase by vitamin b(6) compounds.

The vitamin B(6) compounds pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and pyridoxamine 5'-phosphate (PMP) inhibited the diphenolase activity of mushroom tyrosinase. PM showed the highest inhibition; the control activity was inhibited by 38% at 1.5 mM. Each PL, PN, and PMP showed about 30% inhibition at the same concentration. Lineweaver-Burk plots showed that PM and PN were mixed-type inhibitors with K(I) values of 4.3 and 5.2 mM, respectively. Because PM and PN cannot form a Schiff base with a primary amino group of the enzyme, their inhibition is not attributable to the formation of the Schiff base. Alternatively, their quenching function of reactive oxygen species (ROS) was postulated to be responsible for the inhibition. Thus, the inhibitory effect of ROS was examined. The representative singlet oxygen quenchers l-histidine, sodium azide, Trolox, and anthracene-9,10-dipropionic acid (AAP) inhibited the activity. The specific scavenger of superoxide, proxyl fluorescamine, also inhibited the activity. The scavengers of hydroxyl radical, d-mannitol and dimethyl sulfoxide, showed no inhibition. The fluorescence of AAP was decayed during the diphenolase reaction, and PM inhibited the decay. AAP was also a mixed-type inhibitor. The results showed that the vitamin B(6) compounds inhibited the diphenolase activity by quenching ROS (probably singlet oxygen) generated during some reaction step of the diphenolase reaction.