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J. Biol. Chem., Vol. 283, Issue 36, 25027-25035, September 5, 2008

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Structure and Mechanism of GumK, a Membrane-associated Glucuronosyltransferase^*

From the Laboratory of Bacterial Genetics, Fundación Instituto Leloir, IIBBA-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires C1405BWE, Argentina

Xanthomonas campestris GumK (β-1,2-glucuronosyltransferase) is a 44-kDa membrane-associated protein that is involved in the biosynthesis of xanthan, an exopolysaccharide crucial for this bacterium's phytopathogenicity. Xanthan also has many important industrial applications. The GumK enzyme is the founding member of the glycosyltransferase family 70 of carbohydrate-active enzymes, which is composed of bacterial glycosyltransferases involved in exopolysaccharide synthesis. No x-ray structures have been reported for this family. To better understand the mechanism of action of the bacterial glycosyltransferases in this family, the x-ray crystal structure of apo-GumK was solved at 1.9Å resolution. The enzyme has two well defined Rossmann domains with a catalytic cleft between them, which is a typical feature of the glycosyltransferase B superfamily. Additionally, the crystal structure of GumK complexed with UDP was solved at 2.28Å resolution. We identified a number of catalytically important residues, including Asp¹⁵⁷, which serves as the general base in the transfer reaction. Residues Met²³¹, Met²⁷³, Glu²⁷², Tyr²⁹², Met³⁰⁶, Lys³⁰⁷, and Gln³¹⁰ interact with UDP, and mutation of these residues affected protein activity both in vitro and in vivo. The biological and structural data reported here shed light on the molecular basis for donor and acceptor selectivity in this glycosyltransferase family. These results also provide a rationale to obtain new polysaccharides by varying residues in the conserved /β/ structural motif of GumK.

Received for publication, February 14, 2008 , and in revised form, June 30, 2008.

The atomic coordinates and structure factors (codes 2HY7, 2Q6V, 3CUY, and 3CV3) 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 Agencia Nacional de Promoción Científica y Tecnológica Grant PICT 1-11703 and Universidad de Buenos Aires (Argentina) Grant UBACyT X-193. 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 1 and Figs. 1 and 2.

¹ Máximo Barreras is the recipient of a doctoral fellowship from Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Argentina.

² Silvina R. Salinas is the recipient of a doctoral fellowship from Agencia Nacional de Promoción Cientifica y Tecnológica.

³ Patricia L. Abdian is a member of CONICET.

⁴ To whom correspondence should be addressed: Fundación Instituto Leloir. Av. Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina. Fax: 54-5238-7501; E-mail: Lielpi{at}leloir.org.ar.

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