Isolation and Biochemical Characterization of Hypophosphite/ 2-Oxoglutarate Dioxygenase

Abstract

The htxA gene is required for the oxidation of hypophosphite in Pseudomonas stutzeri WM88 (Metcalf, W. W., and Wolfe, R. S. (1998) J. Bacteriol. 180, 5547-5558). Amino acid sequence comparisons suggest that hypophosphite:2-oxoglutarate dioxygenase (HtxA) is a novel member of the 2-oxoglutarate-dependent dioxygenase enzyme family. To provide experimental support for this hypothesis, HtxA was overproduced in Escherichia coli and purified to apparent homogeneity. Recombinant HtxA is identical to the native enzyme based on amino terminus sequencing and mass spectral analysis, and it catalyzes the oxidation of hypophosphite to phosphite in a process strictly dependent on 2-oxoglutarate, ferrous ions, and oxygen. Succinate and phosphite are stoichiometrically produced, indicating a strict coupling of the reaction. Size exclusion analysis suggests that HtxA is active as a homodimer, and maximal activity is observed at pH 7.0 and at 27°C. The apparent K m values for hypophosphite and 2-oxoglutarate were 0.58 ؎ 0.04 mM and 10.6 ؎ 1.4 M, respectively. V max and k cat values were determined to be 10.9 ؎ 0.30 mol min ؊1 mg ؊1 and 355 min ؊1 , respectively. 2-Oxoadipate and pyruvate substitute poorly for 2-oxoglutarate as a cosubstrate. The highest specific activity is observed with hypophosphite as substrate, but HtxA is also able to oxidize formate and arsenite at significant rates. The substrate analog inhibitors, formate and nitrate, significantly reduce HtxA activity.The current view of phosphorus metabolism dictates that, unlike other elements essential for growth, phosphorus does not undergo a biologically catalyzed oxidation-reduction cycle in nature. The biochemistry involving this essential and often limiting nutrient is typically thought to be restricted to the formation and hydrolysis of phosphate esters, in which phosphorus exists in its most oxidized state (P 5ϩ ). However, the number of microorganisms capable of using reduced phosphorus compounds as the sole source of phosphorus or able to synthesize reduced phosphorus compounds clearly demonstrates that this is not the case (1-5). Although microbial metabolism of reduced phosphorus compounds has been documented in the literature for several decades, it has remained unexplored in any detail on either the biochemical or genetic levels until recently. This is especially true with respect to the microbial oxidation of the reduced P i compounds, phosphite (P 3ϩ ) and hypophosphite (P ϩ ). Microbial growth on hypophosphite or phosphite as the sole source of phosphorus has been reported in such microorganism as Escherichia coli, Bacillus spp., Pseudomonas fluorescens, Klebsiella aerogenes, and Erwinia spp. (2, 4, 6 -9); however, little is known about the biochemistry of hypophosphite or phosphite oxidation in these organisms. In P. fluorescens, activity of partially purified phosphorus-oxidizing enzyme was demonstrated to be NAD ϩ -dependent and specific for phosphite; however, a more detailed analysis was not completed (10). Similarly, hypophosphite oxida...

Genetic Diversity and Horizontal Transfer of Genes Involved in Oxidation of Reduced Phosphorus Compounds by Alcaligenes faecalis WM2072

“…These data indicate that there is an alternate phosphate-oxidizing activity in A. faecalis, which, based on the lack of growth by ptxD htxA double mutants, can be attributed to the HtxA protein. Consistent with this result, HtxA from P. stutzeri, which is identical to A. faecalis HtxA, has been shown to be capable of poorly oxidizing phosphite in vitro (23). The apparent contradiction in the P. stutzeri in vivo and in vitro data (i.e., HtxA catalyzes phosphite oxidation in vitro, but ptxABCDE deletion mutants cannot perform this reaction in vivo even though they are htxA ϩ ) may be explained by differences in the uptake systems for reduced P compounds in the two organisms.…”

“…The P. stutzeri htxD gene is longer than the A. faecalis htxD gene; however, the genes are nearly identical (ϳ98% nucleotide identity) in the shared region. Given that the htxA product of P. stutzeri has been biochemically proven to be a hypophosphite:2-oxogluarate dioxygenase (23), there can be no doubt that the identical A. faecalis HtxA protein also possesses this property. HtxBCD probably form a phosphate-hypophosphite transporter (see below).…”

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

“…Thus, despite the fact that both organisms have active phosphatases, neither can oxidize Pt at rates sufficient to support growth. The P. stutzeri WM3617 (⌬ptxA-htxP, ⌬phn) contains mutations that eliminate the two characterized Pt oxidation pathways of this organism but that do not effect phosphatase expression (9,10). (B) BAP (E. coli alkaline phosphatase), SAP (shrimp alkaline phosphatase; Roche Applied Science, Mannheim, Germany) and CIP (calf intestinal phosphatase; Sigma) were assayed for Pt oxidation as described above.…”

“…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).…”

Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

“…Although the actual purpose of the enzyme is still not fully understood, the simple hypothesis, that it is a means for the bacteria to generate free phosphate groups for uptake and use (20). In bacteria, alkaline phosphatase is located in the periplasmic space.…”