CRYSTAL STRUCTURE OF GUMK, A BETA-GLUCURONOSYLTRANSFERASE INVOLVED IN XANTHAN BIOSYNTHESIS

BARRERAS, MÁXIMO; KAMPEL, MATIAS A. AND IELPI, LUIS

Ciudad de Rosario, Pcia de Santa Fé, Argentina

Congreso; XXXXII Reunión Anual de la SAIB; 2006

Prokaryotic glycosyltransferases are enzymes involved in the synthesis of polysaccharides. The bacterial glucuronosyltransferase GumK is involved in the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl, a intermediate step in the synthesis of xanthan, an exopolysaccharide produced by Xanthomonas campestris. Here we describe the molecular structure of this membrane-associated protein at 1.9 angstrom resolution. The molecule has overall dimensions of 45 x 45 x 60 angstrom. The structure of this enzyme shows a Rossmann-type fold, consisting primarily of alpha/beta/alpha sandwiches. It is arranged in 2 globular domains connected by a linker loop. This loop is the bottom of a deep cleft that separates both domains. The cleft has a maximum width of 25 angstrom and a depth of 15 angstrom.  Putative catalytic aminoacids, which are now being mutagenized, lie on loops on the inner surface of this cleft with their reactive side-chains pointing inside it. To study the binding of substrates we performed co-crystallization and soaking of crystals in crystallization solutions containing the donor or the acceptor substrate. In 2Fo-Fc electronic density maps we found the position were a portion of the donor substrate binds. We describe the molecular contacts and interactions involved in this binding, as well as the possible implications for catalysis.