Enzymes of vitamin B6 degradation. Purification and properties of pyridoxine 5'-dehydrogenase (oxidase).

Isolation and identification of a soil bacterium, Arthrobacter Cr-7, that grows with pyridoxine as a sole source of carbon and nitrogen are described. An inducible pyridoxine 5'-dehydrogenase (oxidase) (EC 1.1.99.9) that catalyzes conversion of pyridoxine to isopyridoxal, Pyridoxine + X----isopyridoxal + XH2, the first step in utilization of pyridoxine as a growth substrate by this organism, was purified about 520-fold to homogeneity. The enzyme (Mr = 112,000) is a dimer of probably identical subunits and requires FAD (KD(app) = 0.24 microM) as coenzyme. It oxidizes only pyridoxine (Km = 0.18 mM) and a few related compounds (4-deoxypyridoxine, pyridoxamine, pyridoxal) that contain a free 5-CH2OH group and utilizes oxygen (Km = 0.28 mM), 2,6-dichloroindophenol, or quinones, but not NAD+ or NADP+, as hydrogen acceptors (X in reaction above). With pyridoxine and oxygen as substrates, the enzyme has a broad pH optimum (from pH 7.0 to 8.3), a Vmax of 11.9 mumol X min-1 X mg-1, and a turnover number of 22 s-1 at 25 degrees C. The enzyme is strongly inhibited by sulfhydryl reagents. Except for its substrate specificity, these properties do not differ greatly from those of other flavin-dependent oxidases.