PcxL and HpxL Are Flavin-dependent, Oxime-forming -oxidases in Phosphonocystoximic Acid Biosynthesis in

Several oxime-containing small molecules have useful properties, including antimicrobial, insecticidal, anticancer, and immunosuppressive activities. Phosphonocystoximate and its hydroxylated congener, hydroxyphosphonocystoximate, are recently discovered oxime-containing natural products produced by sp. NRRL S-481 and NRRL WC-3744, respectively. The biosynthetic pathways for these two compounds are proposed to diverge at an early step in which 2-aminoethylphosphonate (2AEPn) is converted to ()-1-hydroxy-2-aminoethylphosphonate (()-1H2AEPn) in but not in sp. NRRL S-481). Subsequent installation of the oxime moiety into either 2AEPn or ()-1H2AEPn is predicted to be catalyzed by PcxL or HpxL from sp. NRRL S-481 and NRRL WC-3744, respectively, whose sequence and predicted structural characteristics suggest they are unusual -oxidases. Here, we show that recombinant PcxL and HpxL catalyze the FAD- and NADPH-dependent oxidation of 2AEPn and 1H2AEPn, producing a mixture of the respective aldoximes and nitrosylated phosphonic acid products. Measurements of catalytic efficiency indicated that PcxL has almost an equal preference for 2AEPn and ()-1H2AEPn. 2AEPn was turned over at a 10-fold higher rate than ()-1H2AEPn under saturating conditions, resulting in a similar but slightly lower / We observed that ()-1H2AEPn is a relatively poor substrate for PcxL but is clearly the preferred substrate for HpxL, consistent with the proposed biosynthetic pathway in HpxL also used both 2AEPn and ()-1H2AEPn, with the latter inhibiting HpxL at high concentrations. Bioinformatic analysis indicated that PcxL and HpxL are members of a new class of oxime-forming -oxidases that are broadly dispersed among bacteria.