NAD PHOSPHITE OXIDOREDUCTASE A NOVEL CATALYST FROM BACTERIA FOR REGENERATION OF NAD(P)H

摘要

A gene encoding an enzyme required for operation of a novel biochemical pathway for oxidation of the reduced phosphorus (P) compound phosphite was cloned from Pseudomonas and also found in other bacteria. The enzyme (designated PtxD) was overproduced in the host Escherichia coli by use of a recombinant system and purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. Mechanistic studies indicate stereoselective transfer of hydride from phosphite to the Re-face of NAD<+> with observed steady-state kinetic isotope effects of 2.1 on Vmax and 1.8 on Vmax/Km. The novel enzyme is useful for methods requiring regenerating the cofactor NADH, for use in synthetic oxidoreductases, and to synthesize chiral compounds, complex carbohydrates, and isotopically-labelled compounds.