Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Abstract

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 K m values of 53.1 ؎ 6.7 M and 54.6 ؎ 6.7 M, for phosphite and NAD, respectively. V max and k cat were determined to be 12.2 ؎ 0.3 mol min ؊1 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.Phosphorus is widely reported to be a redox conservative element in biological systems, with the sum total of phosphorus biochemistry consisting of the formation and hydrolysis of phosphate-ester bonds. These reports imply that reduced phosphorus compounds are not important in living systems and that enzymatically catalyzed redox reactions of phosphorus compounds do not occur; however, an increasing body of evidence indicates that this is not the case. Although it is true that inorganic phosphate (P valence ϩ5) is the principal form of phosphorus in living systems and that phosphate-esters play a critical role in phosphate biochemistry, it is now clear that reduced phosphorus compounds of both natural and xenobiotic origin play important roles in numerous biological systems. Accordingly, many organisms have been shown to possess metabolic pathways for reduction of phosphate to a variety of reduced phosphorus compounds (1-3); others have been shown to possess metabolic pathways for oxidation of reduced phosphorus compounds (4 -9). Among the most striking of these is a recently isolated sulfate-reducing bacterium that obtains all of the energy it requires for growth from the oxidation of phosphite (ϩ3 valence) to phosphate (10).Unfortunately, detailed studies examining the mechanisms of biological phosphorus oxidation and reduction are scarce. This is particularly true with regard to the biochemical characterization of enzymes involved in reduced p...

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase

“…Pt dehydrogenase (PtxD) had been the only in vitrocharacterized enzyme shown to catalyze Pt oxidation (11). Although the details of the BAP reaction remain to be elucidated, the chemical mechanisms of the two enzymes are clearly distinct.…”

“…11, with the additional modification of omitting the addition of sodium citrate. Activity was determined by assaying the level of phosphate present at sequential time points over a 30-min reaction in 50 mM Mops buffer (pH 7.0) with 10 mM sodium Pt (pH 7.0) at 37°C.…”

“…For example, Pseudomonas stutzeri is capable of oxidizing hypophosphite (P valence, ϩ1) to phosphate by means of a Pt (P valence, ϩ3) intermediate (9). The following two enzymes catalyze these reactions: 2-oxoglutarate͞hypophosphite dioxygenase catalyzes the oxidation of hypo-Pt to Pt (10), and Pt͞NAD oxidoreductase catalyzes the oxidation of Pt to phosphate (11). The latter reaction is the most thermodynamically favorable reduction of NAD that is known, and this trait makes this enzyme particularly useful as a cofactor-regenerating catalyst for enzyme-based synthetic strategies (12).…”

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

“…1). Using such a cycling assay has the advantage that a relatively low NAD + concentration can be used, preventing accumulation of NADH, which is a strong inhibitor of PtxD [19].…”

“…It is therefore likely that chemistry within the extracts produces an inhibitory factor. Known inhibitors of PtxD activity are sulfite and arsenite [19]. It is possible that sulfite is released through degradation of sulfur containing metabolites such as cysteine, glutathione or glucosinolates, which are a major class of secondary metabolite in plants.…”