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J. Biol. Chem., Vol. 283, Issue 23, 15724-15731, June 6, 2008

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A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana

DOMAIN SWAPPING TO INTRODUCE NEW ACTIVITIES^*

Adam M. Cartwright¹, Eng-Kiat Lim, Colin Kleanthous, and Dianna J. Bowles²

From the Centre for Novel Agricultural Products and the Department of Biology, University of York, York YO10 5DD, United Kingdom

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4 '-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4 '-O-glucosides through the use of whole-cell biocatalysis.

Received for publication, March 12, 2008 , and in revised form, March 28, 2008.

^* This work was supported in part by the Garfield Weston Foundation for the Centre for Novel Agricultural Products. 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–S4 and Tables S1 and S2.

¹ Supported by a Biotechnology and Biological Sciences Research Council studentship.

² To whom correspondence should be addressed. Tel.: 44-1904-328770; Fax: 44-1904-328772; E-mail: djb32{at}york.ac.uk.

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