Role of the Tyr270 residue in 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti.

The flavoenzyme 2-Methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) catalyzes the cleavage of the pyridine ring of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) in the presence of NADH, molecular oxygen, and water. MHPCO also catalyzes the NADH oxidation reaction uncoupled with ring opening in the absence of MHPC (the basal activity). The enzyme shows activity toward not only MHPC but also 5-hydroxynicotinic acid (5HN) and 5-pyridoxic acid (5PA). The reaction rate toward 5PA is extremely low (5% of the activity toward MHPC or 5HN). We determined the crystal structures of MHPCO without substrate and the MHPCO/5HN and MHPCO/5PA complexes, together with a Y270F mutant without substrate and its 5HN complex. The Tyr270 residue was located in the active site and formed hydrogen bonds between the Oη and water molecules to make the active site hydrophilic. Although Tyr270 took a fixed conformation in the structures of the MHPCO and MHPCO/5HN complex, it took two conformations in its 5PA complex, accompanied by two conformations of the bound 5PA. In the wild-type (WT) enzyme, the turnover number of the ring-opening activity was 6800 times that of the basal activity (1300 and 0.19 s-1, respectively), whereas no such difference was observed in the Y270F (19 and 7.4 s-1) or Y270A (0.05 and 0.84 s-1) mutants. In the Y270F/5HN complex, the substrate bound ∼1 Å farther away than in the WT enzyme. These results revealed that Tyr270 is essential to maintain the WT conformation, which in turn enhances the coupling of the NADH oxidation with the ring-opening reaction.

Crystal structure of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD⁺-dependent dismutase from Mesorhizobium loti.

5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD(+) and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPCDH are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active site residues in the reaction mechanism are discussed.

Structure of 4-pyridoxolactonase from Mesorhizobium loti.

4-Pyridoxolactonase from Mesorhizobium loti catalyzes the zinc-dependent lactone-ring hydrolysis of 4-pyridoxolactone (4PAL) to 4-pyridoxic acid (4PA) in vitamin B6 degradation pathway I. The crystal structures of 4-pyridoxolactonase and its complex with 5-pyridoxolactone (5PAL; the competitive inhibitor) were determined. The overall structure was an αß/ßα sandwich fold, and two zinc ions were coordinated. This strongly suggested that the enzyme belongs to subclass B3 of the class B ß-lactamases. In the complex structure, the carbonyl group of 5PAL pointed away from the active site, revealing why it acts as a competitive inhibitor. Based on docking simulation with 4PAL, 4PA and a reaction intermediate, 4-pyridoxolactonase probably catalyzes the reaction through a subclass B2-like mechanism, not the subclass B3 mechanism.

Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti.

Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His6-tag and PNOX-pyridoxamine (PM) complex were determined at 2.2Å and at 2.1Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1Å from the N4' atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4'-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4' atom in PM is located at 3.2Å, and the hydride ion from the C4' atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.

Contents of all forms of vitamin B6, pyridoxine-ß-glucoside and 4-pyridoxic acid in mature milk of Japanese women according to 4-pyridoxolactone-conversion high performance liquid chromatography.

The contents of six vitamin B6 forms, pyridoxine-ß-glucoside, and 4-pyridoxic acid in mature milk of 20 Japanese lactating women consuming ordinary Japanese foods were determined by a 4-pyridoxolactone-conversion HPLC method. These compounds were determined with the average recovery rate of 83.9% or more. The average total content of vitamin B6 forms was 1.01 ± 0.32 (µmol/L). Pyridoxal and pyridoxal 5'-phosphate were found in all of the samples, and their average contents were 0.71 ± 0.28 (µmol/L) and 0.16 ± 0.07 (µmol/L), respectively. Pyridoxamine, pyridoxine, pyridoxamine 5'-phosphate, pyridoxine 5'-phosphate, and pyridoxine-ß-glucoside were found in 15, 14, 13, 9, and 7 samples, respectively. The presence of pyridoxine 5'-phosphate was for the first time found in human milk. A method for the determination of 4-pyridoxic acid, which is the excretion form of vitamin B6, was modified to quantitate it by isocratic HPLC. 4-Pyridoxic acid was found in all samples, and its average content was 0.094 ± 0.040 (µmol/L), which was only 12% of its content in cow (Holstein) milk. The total content of vitamin B6 forms, and predominant presence of pyridoxal among other vitamin B6 forms in the Japanese women's milk samples shared similar characteristics with American women's milk samples.

Abnormality in expression levels of gluconeogenesis-related genes by high-dose supplementation with pyridoxamine in mice.

Pyridoxamine supplementation caused the alteration of the expression of genes encoding six gluconeogenesis-related proteins. The expression levels of phosphoenolpyruvate carboxykinase, pyruvate kinase, and pyruvate dehydrogenase kinase 4 in the pyridoxamine-supplemented mice were higher than those in the control mice. In contrast, the pyridoxamine supplementation caused lower expression levels of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, carbohydrate response element-binding protein, glucocorticoid receptor, and glucose-6-phosphatase. The pyridoxamine-supplemented mice showed significantly low glucose clearance in a glucose tolerance test, but they showed no symptoms of diabetes, which was estimated according to the levels of hemoglobin A1c and blood glucose. Pyruvate challenge testing suggested that pyridoxamine supplementation enhanced gluconeogenic activity from pyruvate. The results showed that a high-dose of pyridoxamine may require a careful inquiry concerning its validity.

All-enzymatic HPLC method for determination of individual and total contents of vitamin B(6) in foods.

BACKGROUND: There is a need for a reliable and accurate method for quantification of each of the seven individual vitamin B(6) compounds including pyridoxine-ß-glucoside in foods. OBJECTIVE: To determine pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP), pyridoxine 5'-phosphate (PNP), and pyridoxine-ß-glucoside (PNG) in foods. DESIGN: By specific enzymatic treatment, each of the seven vitamin B(6) compounds was all converted into 4-pyridoxolactone, which is a highly fluorescent compound. In total, seven separate, enzymatic steps were performed for each sample. Separation and quantification were performed with reversed-phase high performance liquid chromatography (HPLC) coupled with fluorescence detection. For each sample type the result was corrected for the recovery based on spiked samples. The method was applied for analyses of chicken liver, chicken white meat, egg yolk, egg white, dried anchovy, carrots, and garlic. RESULTS: The recovery varied from 14 to 114% in chicken liver, chicken white meat, egg yolk, egg white, dried anchovy, carrot, and garlic. Each food showed a characteristic distribution of the seven vitamin B(6) compounds. The PNG was only found in low amounts; that is, 17-29nmol vitamin B(6)/g in the plant-derived foods, carrot and garlic. Only egg white showed a lower content, 3nmol/g. Overall the content in chicken liver, chicken white meat, and egg yolk had a total content of vitamin B(6) between 42 and 51nmol/g. Both PM and PMP were high in the chicken liver. In contrast, PL and PLP were high in the chicken white meat. The main vitamin B(6) in the egg yolk was PLP. The dried anchovy contained high amounts of PLP and PMP and a total content of 144nmol/g. CONCLUSIONS: The enzymatic-based HPLC method was applied for the determination of seven vitamin B6 compounds in foods. Their distribution in the foods varied significantly.

The mll6786 gene encodes a repressor protein controlling the degradation pathway for vitamin B6 in Mesorhizobium loti.

Pyridoxine is converted to succinic semialdehyde, acetate, ammonia and CO(2) through the actions of eight enzymes. The genes encoding the enzymes occur as a cluster on the chromosomal DNA of Mesorhizobium loti, a symbiotic nitrogen-fixing bacterium. Here, it was found that disruption of the mll6786 gene, which is located between the genes encoding the first and eighth enzymes of the pathway, caused constitutive expression of the eight enzymes. The protein encoded by the mll6786 gene is a member of the GntR family and is designated as PyrR. PyrR comprises 223 amino acid residues and is a dimeric protein with a subunit molecular mass of 25 kDa. The purified PyrR with a C-terminal His(6) -tag could bind to an intergenic 67-bp DNA region, which contains a palindrome sequence and a deduced promoter sequence, between the mll6786 and mlr6787 genes, encoding PyrR and AAMS amidohydrolase, respectively.

Crystallization and preliminary X-ray analysis of pyridoxine 4-oxidase, the first enzyme in pyridoxine degradation pathway I.

Vitamin B(6)-degradation pathway I has recently been identified in Mesorhizobium loti MAFF303099. Pyridoxine 4-oxidase, an FAD-dependent enzyme, is the first enzyme in this pathway and catalyzes the irreversible oxidation of pyridoxine to pyridoxal. The enzyme was overexpressed in Escherichia coli with a His(6) tag and purified. The recombinant enzyme was crystallized at 277 K by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. The crystal, which belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 62.38, b = 79.44, c = 136.43 Å, diffracted to 2.2 Å resolution. The calculated V(M) value (3.19 Å(3) Da(-1)) suggested that the asymmetric unit contained one molecule.

Individual vitamin B6 contents in selected Japanese sushi toppings.

The contents of six natural vitamin B(6) forms in popular Japanese sushi toppings were determined by a 4-pyridoxolactone-conversion HPLC method. The half-baked bonito exhibited the highest total vitamin B(6) content and the northern shrimp sashimi the lowest. Pyridoxal 5'-phosphate plus pyridoxal was predominant in nine samples, and pyridoxamine 5'-phosphate plus pyridoxamine in two other samples. Pyridoxine 5'-phosphate plus pyridoxine was minor. The raw meats (sashimi) of fatty seawater fishes contain a lot of pyridoxal 5'-phosphate and/or pyridoxamine 5'-phosphate. Five portions of sushi with 20 g of fatty seawater sashimi toppings would supply with vitamin B(6) recommended by the Japanese Recommended Daily Allowance.

The crystal structure of SDR-type pyridoxal 4-dehydrogenase of Mesorhizobium loti.

Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B(6) compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD(+)-bound enzyme.

Development of simultaneous enzymatic assay method for all six individual vitamin B6 forms and pyridoxine-beta-glucoside.

A method for determining all of the six natural vitamin B(6) compounds and pyridoxine-beta-glucoside in urine from humans consuming their usual diet was developed. These compounds were specifically converted with 5 enzymes into a high fluorescent 4-pyridoxolactone which was supersensitively determined by an isocratic HPLC. All of the compounds in urine from humans consuming their usual diets were for the first time determined together. The preparation procedure for urine samples was easy without HCl-hydrolysis, and the enzyme reactions took only 2 or 3 h. It required only 5 microL of the urine sample for analysis of one of the compounds. Urine samples from five young Japanese males consuming their usual diet contained pyridoxal, pyridoxamine, and pyridoxine-beta-glucoside but not pyridoxine or phosphoester forms. The contents of 4-pyridoxic acid and pyridoxal correlate well with a correlation coefficient of 0.98. On the other hand, the content of pyridoxamine did not correlate with that of 4-pyridoxic acid.

Crystallization and preliminary X-ray analysis of SDR-type pyridoxal dehydrogenase from Mesorhizobium loti.

Pyridoxal 4-dehydrogenase from Mesorhizobium loti MAFF303099 was overexpressed in Escherichia coli. The recombinant selenomethionine-substituted enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 4000 as precipitant. Crystals grew in the presence of 0.45 mM NAD(+). The crystals diffracted to 2.9 A resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 86.20, b = 51.11, c = 91.73 A, beta = 89.36 degrees. The calculated V(M) values suggested that the asymmetric unit contained four molecules.

Esmond E. Snell--the pathfinder of B vitamins and cofactors.

Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

Topology of 4-Pyridoxic acid dehydrogenase in transformed Escherichia coli cells.

The topology of 4-pyridoxic acid dehydrogenase in the Escherichia coli cell membrane was examined with transformed E. coli cells overexpressing the enzyme from Mesorhizobium loti. The recombinant enzymes with a His(6)-tag either in the N-terminal region or at the C-terminus were localized on the E. coli cell membrane like the wild-type enzyme without a His(6)-tag. The His(6)-tags were labelled with Ni-NTA AP conjugate only when the E. coli protoplast cells were broken. The membrane-bound enzyme in the intact protoplast cells was not digested by trypsin, although the one in the gently broken protoplast cells was almost totally digested. Thus, 4-pyridoxic acid dehydrogenase was an integral monotopic protein, protruding into a cytoplasm side from the bacterial membrane. The deletion or mutation of a deduced transmembrane segment in 4-pyridoxic acid dehydrogenase made it an inclusion body, and the enzyme protein was not found in the E. coli cell membrane. Thus, it was suggested that the intact deduced transmembrane segment was necessary for 4-pyridoxic acid dehydrogenase to be localized on the bacterial cell membrane.

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.

Crystallization and preliminary X-ray analysis of AAMS amidohydrolase, the final enzyme in degradation pathway I of pyridoxine.

alpha-(N-Acetylaminomethylene)succinic acid (AAMS) amidohydrolase from Mesorhizobium loti MAFF303099, which is involved in a degradation pathway of vitamin B(6) and catalyzes the degradation of AAMS to acetic acid, ammonia, carbon dioxide and succinic semialdehyde, has been overexpressed in Escherichia coli. To elucidate the reaction mechanism based on the tertiary structure, the recombinant enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as precipitant. A crystal of the enzyme belonged to the monoclinic space group C2, with unit-cell parameters a = 393.2, b = 58.3, c = 98.9 A, beta = 103.4 degrees , and diffraction data were collected to 2.7 A resolution. The V(M) value and calculation of the self-rotation function suggested that three dimers with a threefold symmetry were possibly present in the asymmetric unit.

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).