KINETIC AND COMPLEMENTATION STUDIES ON NUCLEOTIDE-BINDING MUTANTS OF GLYCOSYLTRANSFERASE GUMK

MÁXIMO BARRERAS; SILVINA R. SALINAS; LUIS IELPI

Mar del Plata, Argentina

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

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-a-1,3-glucose-a-1,4-glucose-P-P-polyisoprenyl, an intermediate step in the synthesis of xanthan, an exopolysaccharide produced by Xanthomonas campestris. Based on previous structural data from our laboratory we studied the molecular contacts that bind the donor substrate to the enzyme, performing site-directed mutagenesis on the residues involved. Besides, based on the proposed catalytic mechanism for inverting glycosyltransferases, we mutated individual acidic residues whose positions in the crystal structure make them candidates to be the catalytic residue. Mutated proteins were purified and the kinetic parameters for each mutant were analyzed trough an enzymatic assay using radioactive donor substrate. Also, the mutant ORFs were cloned in the wide-host plasmid pBBRprom and the effect of mutations on the activity of the enzyme was assessed in vivo trough the quantification of xanthan production in complementation assays on gumK- mutant strain XcK. Finally, the gathering of biochemical and structural data has allowed us to postulate aspartate 157 as the catalytic residue, responsible for the nucleophilic attack on the acceptor substrate.