The plmS 2 -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 theplmS 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 theplmS 2 ORF downstream of theermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbpplmS 2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein inStreptomyces coelicolor . The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 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 PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM andk 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.