Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461

Abstract

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis 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, Henrissat (1997) Biochem. J. 326, 929–939]. Sequence similarity studies suggest 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 (GelKNH2) and -CO2H (GelKCOOH) halves were overexpressed in Escherichia coli. GelK and GelKNH2 were present in both the soluble and membrane fractions of E. coli, whereas GelKCOOH was only present in the soluble fraction. GelK catalysed the transfer of [14C]glucuronic acid from UDP-[14C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by β-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PPi-lipid moiety is needed for enzymic activity. Recombinant GelKNH2 and GelKCOOH did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Two Proteins Homologous to the N- and C-terminal Domains of the Bacterial Glycosyltransferase Murg Are Required for the Second Step of Dolichyl-linked Oligosaccharide Synthesis in Saccharomyces cerevisiae

“…In one experiment where the N-and C-terminal domains of the former type of enzyme (Fig. 1, Gelk) have been dissociated and expressed separately there was no evidence for transferase activity when the domains were assayed either independently or as an equimolar mixture (29). Nevertheless, it is possible that the two domains must be expressed together to generate the active complex, but to the best of our knowledge this experiment has not been performed on this type of enzyme.…”

“…second step of LLO biosynthesis in S. cerevisiae correspond to the two protein domains that have been shown to be required for UDP-sugar:undecaprenyl pyrophosphoryl monosaccharide glycosyltransferases involved in bacterial cell wall glycoconjugate biosynthesis pathways (9,26,29). These two protein domains are usually formed by a single polypeptide chain, but in a few instances the domains correspond to separate polypeptide chains encoded by different genes (26).…”

Biosynthesis of Lipid-linked Oligosaccharides in Saccharomyces cerevisiae

“…In this article we have identified and characterized two essential genes, ALG13 and ALG14, necessary for the formation of Alg13p and Alg14p, identified in this work, are evolutionarily conserved proteins in eukaryotic cells and also have a similarity to domains of glycosyltransferase involved in bacterial cell wall glycoconjugate and polysaccharide biosynthesis, respectively (41)(42)(43). For most eukaryotes, the Alg13 and Alg14 proteins are translated from two individual loci found in the genome, the ALG13 and the ALG14 loci, respectively.…”

Functional and Topological Analysis of the Burkholderia cenocepacia Priming Glucosyltransferase BceB, Involved in the Biosynthesis of the Cepacian Exopolysaccharide

“…The reaction was started by the addition of 0.10 Ci of the substrate UDP-[ 14 C]glucose (UDP-[ 14 C]Glc) (NEN Life Science Products). The radiolabeled sugars, covalently linked to the membrane acceptor, were extracted in the lipid fraction and measured as previously described (22). The extracts expressing the BceB protein [C43(pBceB432)] incorporated approximately 10-fold more radiolabeled sugars than extracts prepared from C43(pWH844) control cells (Fig.…”

“…To assess the predicted BceB activity of undecaprenyl-phos-phate glucosyl-1-phosphate transferase (UndPGlcPT), membrane extracts from IPTG-induced E. coli C43(DE3) cells carrying pBceB432 were used as a source of both BceB and membrane acceptor substrate (isoprenoid lipid) as described before (22). Enzyme assays were carried out in a final volume of 100 l containing the following: 50 g of membrane fraction, 50 mM Tris-HCl (pH 8), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 mM MgCl 2 .…”