Structure and Mechanism of GumK, a Membrane-associated GlucuronosyltransferaseS

• Published in: The Journal of biological chemistry 2008-09, Vol.283 (36), p.25027-25035
• Main Authors: Barreras, Máximo, Salinas, Silvina R., Abdian, Patricia L., Kampel, Matías A., Ielpi, Luis
• Format: Article
• Language: English
• Subjects: Glycobiology and Extracellular Matrices
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M801227200

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 Asp 157 , which serves as the general base in the transfer reaction. Residues Met 231 , Met 273 , Glu 272 , Tyr 292 , Met 306 , Lys 307 , and Gln 310 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 α/β/α structural motif of GumK.