Functional Analysis of Cytochrome P450s Involved in Streptovaricin Biosynthesis and Generation of Anti-MRSA Analogues

Abstract

The streptovaricins, chemically related to the rifamycins, are highly effective antibacterial agents particularly against mycobacteria. Herein, a bioassay-guided investigation of Streptomyces spectabilis CCTCC M2017417 has led to the characterization of streptovaricins as potent compounds against methicillin-resistant Staphylococcus aureus (MRSA). We identified the streptovaricin biosynthetic gene cluster from S. spectabilis CCTCC M2017417 based on genomic sequencing and bioinformatic analysis. Targeted in-frame deletion of five cytochrome P450 genes (stvP1-P5) resulted in the identification of four new streptovaricin analogues, and revealed the functions of these genes as follows: stvP1, stvP4 and stvP5 are responsible for the hydroxylation of C-20, Me-24 and C-28, respectively; stvP2 is possibly involved in formation of the methylenedioxy bridge, and stvP3, a conserved gene found in the biosynthetic cluster for naphthalenic ansamycins, might be related to the formation of naphthalene ring. Biochemical verification of the hydroxylase activity of StvP1, StvP4 and StvP5 was performed, and StvP1 showed unexpected biocatalytic specificity and promiscuity. More importantly, anti-MRSA studies of streptovaricins and derivatives revealed significant structure-activity relationships (SARs): the hydroxyl group at C-28 plays a vital role in antibacterial activity; the hydroxyl group at C-20 substantially enhances activity in the absence of the methoxycarbonyl side chain at C-24, which can increase the activity regardless of the presence of a hydroxyl group at C-20; the inner lactone ring between C-21 and C-24 shows a positive effect on activity. This work provides meaningful information on the SARs of streptovaricins, and demonstrates the utility of the engineering of streptovaricins to yield novel anti-MRSA molecules.