Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases.
Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various alpha-1,3 and/or alpha-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases.
Moulis
Claire
C
Laboratoire de Biotechnologies-Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, 135 avenue de Rangueil, 31077 Toulouse Cedex 4, France.
Joucla
Gilles
G
Harrison
David
D
Fabre
Emeline
E
Potocki-Veronese
Gabrielle
G
Monsan
Pierre
P
Remaud-Simeon
Magali
M
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D017931:DNA Primers / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry*; D016695:Glycosyltransferases / Q000737:chemistry*; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000737:chemistry
D017931:DNA Primers; D009844:Oligosaccharides; D013395:Sucrose; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C033837:alternansucrase; C020025:dextransucrase