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J. Biol. Chem., Vol. 279, Issue 31, 32684-32691, July 30, 2004

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The Position of a Key Tyrosine in dTDP-4-Keto-6-deoxy-D-glucose-5-epimerase (EvaD) Alters the Substrate Profile for This RmlC-like Enzyme^*

Alexandra B. Merkel, Louise L. Major, James C. Errey, Michael D. Burkart¶||, Robert A. Field, Christopher T. Walsh¶, and James H. Naismith**

From the Centre for Biomolecular Sciences, The University, St. Andrews, Scotland, KY16 9ST, United Kingdom, the Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, Norfolk, NR4 7TJ, United Kingdom, and the ^¶Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 Å structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

Received for publication, April 13, 2004 , and in revised form, May 24, 2004.

The atomic coordinates and structure factors (codes 1OFN and 1OI6) 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 a Wellcome Trust program grant (to J. H. N.). 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.

^|| Present address: Dept. of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0358.

^** A Biotechnology Biological Sciences Research Council Career Development Fellow. To whom correspondence should be addressed. Tel.: 44-1334-463792; Fax: 44-1334-462595; E-mail: naismith{at}st-and.ac.uk.

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