Isolation and biochemical characterization of hypophosphite/2-oxoglutarate dioxygenase. A novel phosphorus-oxidizing enzyme from Psuedomonas stutzeri WM88.
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 degrees C. The apparent K(m) values for hypophosphite and 2-oxoglutarate were 0.58 +/- 0.04 mm and 10.6 +/- 1.4 microm, respectively. V(max) and k(cat) values were determined to be 10.9 +/- 0.30 micromol 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.
White
Andrea K
AK
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
Metcalf
William W
WW
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D017905:Phosphites / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D002413:Cations, Divalent; D010710:Phosphates; D017905:Phosphites; D011993:Recombinant Fusion Proteins; D010105:Oxygenases