Functional characterization of GumK, a membrane-associated beta-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis.
Xanthomonas campestris is a Gram-negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely used in the industry as thickener and viscosifier. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components have not been examined and no data on glycosyltransferases have been reported. We describe the functional characterization of the gumK gene product, an essential protein for xanthan synthesis. Immunoblots and complementation studies showed that GumK is a 44-kDa protein associated to the membrane fraction. This value corresponds to the expected molecular mass for GumK encoded by an extended open reading frame than proposed from previous genetic data and in X. campestris published complete genome. The protein was expressed in Escherichia coli cells. The purified protein catalyzed the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl with formation of a glucuronic acid-beta-mannose linkage. We examined the acceptor substrate specificity. GumK was unable to use the trisaccharide acceptor freed from the pyrophosphate lipid moiety. Replacement of the natural lipid moiety by phytanyl showed that the catalytic function could proceed with glucuronic acid transfer. These results suggest the enzyme does not show specificity for the lipidic portion of the acceptor. GumK showed diminished activity when tested with 6-O-acetyl-mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl, a putative intermediate in the synthesis of xanthan. This could indicate that acetylation of the internal mannose takes place after the formation of the GumK product.
Barreras
Máximo
M
Fundación Instituto Leloir, University of Buenos Aires and CONICET, Av. Patricias Argentinas 435, (C1405BWE)Buenos Aires, Argentina.
Abdian
Patricia L
PL
Ielpi
Luis
L
D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli; D005816:Genetic Complementation Test; D014453:Glucuronosyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014974:Xanthomonas / Q000166:cytology / Q000201:enzymology*
D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D014453:Glucuronosyltransferase; C002563:xanthan gum