title: Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A.
abstract: Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 μM and 2.0 ± 0.1 μM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 μM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea.
authors: Songhee
authors: S
authors: Han
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea.
authors: Tan-Viet
authors: TV
authors: Pham
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea.
authors: Joo-Hwan
authors: JH
authors: Kim
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea.
authors: Young-Ran
authors: YR
authors: Lim
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea.
authors: Hyoung-Goo
authors: HG
authors: Park
authors: Chonnam National University, School of Biological Sciences and Technology, Gwangju 500-757, Republic of Korea.
authors: Gun-Su
authors: GS
authors: Cha
authors: Chonnam National University, School of Biological Sciences and Technology, Gwangju 500-757, Republic of Korea.
authors: Chul-Ho
authors: CH
authors: Yun
authors: Chung-Ang University, College of Pharmacy, Seoul 156-756, Republic of Korea.
authors: Young-Jin
authors: YJ
authors: Chun
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea. Electronic address: lkang@konkuk.ac.kr.
authors: Lin-Woo
authors: LW
authors: Kang
authors: Konkuk University, Department of Biological Sciences, Seoul 143-701, Republic of Korea. Electronic address: donghak@konkuk.ac.kr.
authors: Donghak
authors: D
authors: Kim
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D008958:Models, Molecular; D009840:Oligomycins / Q000096:biosynthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D009840:Oligomycins; C031004:oligomycin A; D003577:Cytochrome P-450 Enzyme System
keywords: CYP; CYP107W1; Oligomycin; P450; Streptomyces avermitilis; X-ray crystal structure