Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequentia l transfer of sugars, from the appropriate sugar donor to an activated lipi d carrier, by committed glycosyltransferases (GTs). Few studies on the mech anism of action for this type of GT are available. Sphingomonas paucimobili s A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, He nrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies sugge st that GelK consists of two protein modules corresponding to the -NH2 and -CO2H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH2 (GelK(NH2)) and -CO2H (Gel K(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the tr ansfer of [C-14]glucuronic acid from UDP-[C-14]glucuronic acid into a glyco lipid extracted from S. paucimobilis or E. coli, even in the presence of ED TA, and the radioactive sugar was released from the glycolipid by beta -1,4 -glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activ ity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarit ies with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.