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

Abstract

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.

Phosphonates: Their Natural Occurrence and Physiological Role

“…Similar approach was used for the isolation of phosphonocystoximate and its hydroxylated derivative (compounds 48 and 49) [24]. Detailed NMR studies on their biosynthesis, which starts from ciliatine and its analog-compound 9, enabled to confirm the presence of intermediates such as mixtures of the (E)-and (Z)-isomers of corresponding oximes (compounds 50 and 51), substrates for the synthesis of phosphonocystoximate and its hydroxylated derivative [95]. They are formed by the action of specific flavin-dependent, oxime-forming N-oxidases.…”

New frontiers in flavin-dependent monooxygenases

“…The catalytic mechanism of CrmH is believed to involve the double hydroxylation of the substrate, caerulomycin N, to form dihydroxylamine, which then dehydrates and tautomerizes to form the oxime-containing caerulomycin A. Caerulomycin A has a unique 2,2 ′ -bipyridine skeleton and shows attractive potential bioactivities such as antifungal, antiamoebic, and antitumor [62] properties. In Streptomyces species, PcxL and Hpx catalyze similar oxidation reactions of 2-aminoethylphosphonate (2AEPn) and (R)-1H2AEPn, respectively, to corresponding aldoximes, which are further oxidized to nitrosylated phosphonic acid products through multiple steps [66]. Oxime-containing small molecules are reported to have interesting biological properties such as antibacterial, antifungal, insecticidal, antitumor, immunosuppressive, and acetylcholinesterase reactivation activities [66].…”

“…In Streptomyces species, PcxL and Hpx catalyze similar oxidation reactions of 2-aminoethylphosphonate (2AEPn) and (R)-1H2AEPn, respectively, to corresponding aldoximes, which are further oxidized to nitrosylated phosphonic acid products through multiple steps [66]. Oxime-containing small molecules are reported to have interesting biological properties such as antibacterial, antifungal, insecticidal, antitumor, immunosuppressive, and acetylcholinesterase reactivation activities [66].…”

Phosphorus Compounds of Natural Origin: Prebiotic, Stereochemistry, Application

“…Dehydrophos consists of a glycineleucin dipeptide bound to an O-methylated aminovinylphosphonate, its dehydrophosphorylation proceeds analogously to the similar in structure alaphosphine peptide and the Trojan horse peptide dehydrophosphine is imported and cleaved by peptide permeases and peptidases, respectively, to release the alanine racemase inhibitor within the cell (Figure 58) [179,180]. In recent years, a number of P-C antibiotics, possessing antifungal activity: rhizocticins [139,180], phosphonocystoximic acid [181], plumbemycins [139,182], phosphacetamycin [183], have been isolated from Bacillus subtilis and Streptomyces plumbeus. They represent di-and tripeptides containing the C-terminal (Z)-L-2-amino-5-phosphono-3pentenoic acid, a phosphonotreonine mimetic [184], reminiscent of dehydrophos as a broad-spectrum antibiotic that acts against infection of Salmonella [185].…”