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.

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.

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.

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.

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.