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J. Biol. Chem., Vol. 282, Issue 23, 16942-16947, June 8, 2007

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Enhancing the Latent Nucleotide Triphosphate Flexibility of the Glucose-1-phosphate Thymidylyltransferase RmlA^*

Rocco Moretti¹ and Jon S. Thorson^¶2

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, and ^¶University of Wisconsin National Cooperative Drug Discovery Group, School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705

Nucleotidyltransferases are central to nearly all glycosylation-dependent processes and have been used extensively for the chemoenzymatic synthesis of sugar nucleotides. The determination of the NTP specificity of the model thymidylyltransferase RmlA revealed RmlA to utilize all eight naturally occurring NTPs with varying levels of catalytic efficiency, even in the presence of nonnative sugar-1-phosphates. Guided by structural models, active site engineering of RmlA led to alterations of the inherent pyrimidine/purine bias by up to three orders of magnitude. This study sets the stage for engineering single universal nucleotidyltransferases and also provides new catalysts for the synthesis of novel nucleotide diphosphosugars.

Received for publication, March 7, 2007

^* This work was supported, in part, by National Institutes of Health Grants AI552218 and CA84374 (to J. S. T.). 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 Figs. S1 and S2 and Tables S1 and S2.

¹ A National Institutes of Health Molecular Bioscience Training Grant trainee (GM07215).

² An H. I. Romnes fellow. To whom correspondence should be addressed: School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI 53705. Tel.: 608-262-3829; Fax: 608-262-5345; E-mail: jsthorson{at}pharmacy.wisc.edu.

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