The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with Km values of 53.1 +/- 6.7 microm and 54.6 +/- 6.7 microm, for phosphite and NAD, respectively. Vmax and kcat were determined to be 12.2 +/- 0.3 micromol x min(-1) x mg(-1) and 440 min(-1). NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the d-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound.