PcxL and HpxL are flavin-dependent, oxime-forming N -oxidases in phosphonocystoximic acid biosynthesis in Streptomyces .

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 Streptomyces sp. NRRL S-481 and Streptomyces regensis 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 ( S )-1-hydroxy-2-aminoethylphosphonate (( S )-1H2AEPn) in S. regensis but not in Streptomyces sp. NRRL S-481). Subsequent installation of the oxime moiety into either 2AEPn or ( S )-1H2AEPn is predicted to be catalyzed by PcxL or HpxL from Streptomyces sp. NRRL S-481 and S. regensis NRRL WC-3744, respectively, whose sequence and predicted structural characteristics suggest they are unusual N -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 ( R )-1H2AEPn. 2AEPn was turned over at a 10-fold higher rate than ( R )-1H2AEPn under saturating conditions, resulting in a similar but slightly lower k _cat / K _m We observed that ( S )-1H2AEPn is a relatively poor substrate for PcxL but is clearly the preferred substrate for HpxL, consistent with the proposed biosynthetic pathway in S. regensis. HpxL also used both 2AEPn and ( R )-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 N -oxidases that are broadly dispersed among bacteria.
(© 2018 Goettge et al.)