Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88

The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehyd rogenase, PtxD) was cloned into an expression vector and overproduced in Es cherichia coli, The heterologously produced enzyme is indistinguishable fro m the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneit y via a two-step affinity protocol and characterized. The enzyme stoichiome trically produces NADH and phosphate from NAD and phosphite, The reverse re action was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer, PtxD has a high affinity for i ts substrates with K-m values of 53.1 +/- 6.1 muM and 54.6 +/- 6.7 muM, for phosphite and NAD, respectively. V-max and k(cat) were determined to be 12 .2 +/- 0.3 mu mol min(-1) mg(-1) and 440 min(-1). NADP can substitute poorl y for NAD; however, none of the numerous compounds examined were able to su bstitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most con sistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparison s place PtxD as a new member of the D-2-hydroxyacid NAD-dependent dehydroge nases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxid ation of a reduced phosphorus compound.