Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-β-Dmannosyltransferase from Xanthomonas campestris pv. campestris

SILVINA R. SALINAS, MARÍA I. BIANCO, MÁXIMO BARRERAS, AND LUIS IELPI

GLYCOBIOLOGY

OXFORD UNIV PRESS INC

Año: 2011

0959-6658

We describe the first biochemical characterization of the gumI gene product, anessential protein for xanthan polysaccharide synthesis. Cellular fractionationexperiments reveal a protein associated with the membrane fraction, even in theabsence of the other proteins responsible for the synthesis of glycolipid intermediatesand the proteins involved in the polymerization and transport of the xanthan chains. Byalkaline buffer extraction and detergent phase partitioning, GumI was categorized as amonotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized,and purified in an active and stable form using a simple and reproducible two-stepprocedure. The purified recombinant GumI is a non-processive β-mannosyltransferasethat uses GDP-Man as a donor substrate and glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established theoptimal biochemical conditions for GumI enzymatic activity. Sequence analysisrevealed the presence of a conserved domain for glycosyltransferases (GTs) of the GTBsuperfamily and homologous proteins in several prokaryote organisms. Based on thisbiochemical characterization, GumI may represent the founding member of a new GTfamily in the Carbohydrate-Active EnZymes classification.