HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 24, 17486-17500, June 15, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/24/17486    most recent M700863200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chun, Y.-J. Articles by Guengerich, F. P. Search for Related Content PubMed PubMed Citation Articles by Chun, Y.-J. Articles by Guengerich, F. P. Social Bookmarking                      What's this?

Electron Transport Pathway for a Streptomyces Cytochrome P450

CYTOCHROME P450 105D5-CATALYZED FATTY ACID HYDROXYLATION IN STREPTOMYCES COELICOLOR A3(2)^*

Young-Jin Chun¹, Tsutomu Shimada², Raymundo Sanchez-Ponce, Martha V. Martin, Li Lei, Bin Zhao, Steven L. Kelly^¶, Michael R. Waterman, David C. Lamb^¶, and F. Peter Guengerich³

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea, and ^¶Institute of Life Science and School of Medicine, University of Wales, Swansea SA2 8PP, Wales, United Kingdom

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH FDR1 Fdx4 P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C–H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.

Received for publication, January 30, 2007 , and in revised form, April 17, 2007.

^* This work was supported by National Institutes of Health Grants R01 GM069970 (to M. R. W. and F. P. G.), R37 CA090426 (to F. P. G.), T32 ES07028 (to R. S. and F. P. G.), and P30 ES00267 (to F. P. G. and M. R. W.) and the Biological and Biotechnology Science Research Council, Wellcome Trust, and Society for General Microbiology (to D. C. L. and S. L. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S11.

¹ Present address: College of Pharmacy, Chung-Ang University, 221 Huksuk-Dong, Dongjak-Gu, Seoul 156-756, Republic of Korea.

² Present address: Dept. of Chemical Biology, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan.

³ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. D. Sohl, E. M. Isin, R. L. Eoff, G. A. Marsch, D. F. Stec, and F. P. Guengerich Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2: HIGHLY COOPERATIVE PYRENE HYDROXYLATION AND MULTIPHASIC KINETICS OF LIGAND BINDING J. Biol. Chem., March 14, 2008; 283(11): 7293 - 7308. [Abstract] [Full Text] [PDF]