Cloning and Characterization of Pyridoxal Kinase from Geobacillus sp. H6a

Pyridoxal kinase encoded by pdxK gene, is the important key enzyme in the salvage pathway of vitamin [B.sub.6] biosynthesis. The enzyme catalyzes the phosphorylation of the 5' alcohol groups of free form vitamin [B.sub.6] into their 5'-phosphate forms that requires metal ion and ATP. Pyridoxal kinase have been reported in many organisms except in the thermophilic bacterium. Therefore, this study aimed to clone, express and characterize pyridoxal kinase of Geobacillus sp. H6a isolated from the hot spring in the North of Thailand. The GhpdxK gene (810 base pairs) was inserted into pET28a(+) plasmids at restriction site of Ndel and [beta][alpha]mHI and transformed into E.coli BL21(DE3). The expressed pyridoxal kinase of this bacterium exhibits a homodimer, in which each subunit had a molecular mass of about 32 kDa when examined by SDS-PAGE and gel filtration. The enzyme showed maximal activity at 70[degrees]C and at pH 8.0. The expressed enzyme obtained in this study was found to be more active (>50%) in the broad pH range (6.0 - 9.0) than those previously reported. This enzyme prefers [Mg.sup.2+] and also accepts other cations to the less extent. Under optimal conditions, the expressed enzyme has higher affinity toward PN (20 [+ or -] 1.35 [micro]M), while it showed the same affinity to pyridoxal (100 [+ or -] 0.76 [micro]M) and pyridoxamine (100 [+ or -] 1.21 [micro]M). The [K.sub.m] value for ATP and 4-amino-5-hydroxymethyl-2-methylpyridine were 8.99 [+ or -] 1.76 [micro]M and 19 [+ or -] 0.85 [micro]M, respectively. With high activity at high temperature and active in the broad pH range, it could be considered as a potential candidate for future application particularly bioconversion of vitamin [B.sub.6]. Keywords: pyridoxal kinase, salvage, thermophile, vitamin [B.sub.6]