Analysis of membrane-binding region of a bacterial glucuronosyltransferase.

SALINAS, SR; IELPI, L.

San Miguel de Tucumán, Tucumán

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

Current classification of membrane proteins describes proteins thatcross the membrane at least twice (polytopics), once (bitopics), andnot at all (monotopics). The last one is a very particular group andhas not been fully described yet. GumK is a monotopicglycosyltransferase involved in xanthan biosynthesis, transferringa glucuronic acid residue to a lipidic acceptor. Recently, GumKcrystal structure was solved in our laboratory. Structural data showa patch of basic and hydrophobic aminoacids that would beinvolved in both membrane and acceptor substrate binding.Bionformatic simulation supports this hypothesis. To study the roleof this region, four GumK mutants were constructed: B1 (R55N,R58N, K60Q), B3 (R86N), H1 (L56S, M59S), and H2 (L99S,V102T, M106S). Analysis of subcellular localization show that allmutants are membrane-associated. The activity of these mutantswas studied in a gumK- strain (XcK) by complementation assays.B1 and B3 produce similar amounts of xanthan compared to wildtype, H1 drastically reduces xanthan production, and H2 produces50% less xanthan than wild type. These results are the first evidencethat biological activity of this monotopic protein depends deeply onits partial membrane insertion.