The plmS2-encoded cytochrome P450 monooxygenase mediates hydroxylation of phoslactomycin B in Streptomyces sp. strain HK803

Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS(2) open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS(2) ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS(2) ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS(2) converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS(2) was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS(2) (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS(2) (K(m) of 45.3 +/- 9.0 microM and k(cat) of 0.27 +/- 0.04 s(-1)) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.