NOVEL STRUCTURE OF GUMK, AN ENZYME INVOLVED IN THE SYNTHESIS OF A BACTERIAL POLYSACCHARIDE

MAXIMO BARRERAS AND LUIS IELPI.

Puerto Madryn, Provincia de Chubut, Argentina

Congreso; II Reunion de la Asociacion Argentina de Cristalografia.; 2006

Asociacion Argentina de Cristalografia

One of the mayor objectives in our laboratory is to understand at the molecular level glycosyltransferases (GTs) function. So far just a few of these enzymes have been crystallized and their structure solved (Breton C., et al. 2006. Glycobiology 16, 29-37). Despite this, not much is known on how catalysis and/or substrate binding proceeds in this vast group of enzymes. We describe here the molecular structure of GumK, a membrane-associated bacterial GT that catalyzes the transfer of a glucuronic acid residue to produce a polysaccharide secreted by the bacteria Xanthomonas campestris. This enzyme was cloned, overexpressed and purified from an heterologous host using commercial vectors (Barreras M., et al. 2004. Glycobiology 14, 233-241). GumK crystallization conditions were found using the hanging drop vapor diffusion method. Crystals were cryo-cooled in liquid nitrogen to minimize radiation damage. X-ray diffraction data for native and derivative crystals were obtained in the National Synchotron Light Source, Brookhaven National Laboratories, EEUU. (Barreras M, et al. 2006. Acta Cryst. Section F, Volume 62 (9), 880-883). The structure of the enzyme was solved at 1.9 angstroom resolution. Diffraction phase angle information was obtained through a multiwavelenght anomalous dispersion experiment of a crystal that bind Pt atoms after soaking in a solution of K2PtCl4. The solved structure of GumK shows 373 of a total of 400 aminoacids that compose it. The enzyme has overall dimensions of 45 x 45 x 60 angstrom. The asymmetric unit is a monomer of the protein, consisting of 2 globular domains connected by a loop. The domains have a core of beta-sheet arranged aminoacids surrounded by alpha-helices. These domains are separated by a deep cleft, which has a maximum width of 25 angstrom and a depth of 15 angstrom. The catalytic aminoacids, which are now being mutagenized to study their function, most certainly lie on loops on the inner surface of this cleft with their reactive side-chains pointing inside the cleft. 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 were able to find 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. Keywords: glycosyltransferases, GumK, xanthan