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

J. Biol. Chem., Vol. 281, Issue 36, 26289-26297, September 8, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/36/26289    most recent M605478200v1 Alert me when this article is cited 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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sherman, D. H. Articles by Podust, L. M. Search for Related Content PubMed PubMed Citation Articles by Sherman, D. H. Articles by Podust, L. M. Social Bookmarking                      What's this?

The Structural Basis for Substrate Anchoring, Active Site Selectivity, and Product Formation by P450 PikC from Streptomyces venezuelae^*

David H. Sherman¹, Shengying Li, Liudmila V. Yermalitskaya, Youngchang Kim^¶, Jarrod A. Smith, Michael R. Waterman, and Larissa M. Podust²

From the Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, the Department of Biochemistry and Center in Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232, and the ^¶Argonne National Laboratory, Structural Biology Center, Argonne, Illinois, 60439

The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 Å) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 Å)or narbomycin (resolution 1.7 Å). A central feature of the enzyme-substrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3' dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.

Received for publication, June 7, 2006 , and in revised form, June 30, 2006.

The atomic coordinates and structure factors (code 2BVJ, 2C7X, and 2C6H) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants GM37942, GM69970, ES00627 (to M. R. W.), and GM078553 (to D. H. S.), by grants from the Vanderbilt-Meharry Center for AIDS Research, Vanderbilt University Medical Center (VUMC) Discovery Grant Program, and U. S. Civilian Research and Development Foundation (to L. M. P.). 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 two supplemental movies.

¹ To whom correspondence may be addressed. Tel.: 734-615-9907; Fax: 734-615-3641; E-mail: davidhs{at}umich.edu. ² To whom correspondence may be addressed. Tel.: 615-343-0943; Fax: 615-322-4349; E-mail: larissa.m.podust{at}vanderbilt.edu.

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

This article has been cited by other articles:

				W. S. Jung, S. J. Jeong, S. R. Park, C. Y. Choi, B. C. Park, J. W. Park, and Y. J. Yoon Enhanced Heterologous Production of Desosaminyl Macrolides and Their Hydroxylated Derivatives by Overexpression of the pikD Regulatory Gene in Streptomyces venezuelae Appl. Envir. Microbiol., April 1, 2008; 74(7): 1972 - 1979. [Abstract] [Full Text] [PDF]

				H. Ouellet, L. M. Podust, and P. R. O. de Montellano Mycobacterium tuberculosis CYP130: CRYSTAL STRUCTURE, BIOPHYSICAL CHARACTERIZATION, AND INTERACTIONS WITH ANTIFUNGAL AZOLE DRUGS J. Biol. Chem., February 22, 2008; 283(8): 5069 - 5080. [Abstract] [Full Text] [PDF]