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Journal of Bacteriology, December 2005, p. 7970-7976, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.7970-7976.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The plmS₂-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

Mohini S. Ghatge and Kevin A. Reynolds^*

Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219

Received 8 June 2005/ Accepted 20 September 2005

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 plmS₂ 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 plmS₂ ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS₂ ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces 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 (K_m of 45.3 ± 9.0 µM 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.

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* Corresponding author. Present address: Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR 97207-0751. Phone: (503) 725-3886. Fax: (503) 725-9525. E-mail: reynoldsk{at}pdx.edu.

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Journal of Bacteriology, December 2005, p. 7970-7976, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.7970-7976.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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