Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Abstract

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 ␣-1,3 and/or ␣-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. Glucansucrases from Glycoside-Hydrolase (GH)2 -family 70 (EC. 2.4.1.5) are extracellular enzymes produced by lactic acid bacteria of the genus Leuconostoc, Streptococcus, or Lactobacillus (1). From sucrose, they catalyze the synthesis of high molecular weight glucans. They can also produce oligosaccharides or glucoconjugates by a transglucosylation reaction from the sucrose donor to an exogenous acceptor, and this so called "acceptor reaction" occurs at the cost of polymer synthesis (2, 3). An interesting diversity exists in the GH-family 70, where there are enzymes able to synthesize all the types of glucosidic linkages, namely ␣-1,2; ␣-1,3; ␣-1,4; or ␣-1,6 glucosidic bonds. So, depending on the enzyme specificity, a wide range of glucans can be produced, varying in terms of size, structure, degree of branches and spatial arrangements.Primary structures of at least 44 different glucansucrases are now available in GH-family 70.3 With an average predicted molecular mass of more than 160,000 Da, they all show the same organization consisting of a variable region at the N terminus, a conserved catalytic domain, and a C-terminal domain typically containing a series of homologous repeating units. In a number of streptococcal glucansucrases, as well as for the L. mesenteroides NRRL B-512F dextransucrase, the repeats have been demonstrated to play a role in enzyme glucan bindi...

Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes

“…These results show that the glucosyl moiety of sucrose is transferred to the nonreducing end of maltose and panose. Transfer to the nonreducing end is also in agreement with other studies (13,15) as well as with the structure of GTF180-ΔN with a bound maltose, which shows that maltose binds adjacent to subsite −1 with its nonreducing end C6 hydroxyl group pointing towards the catalytic center. Thus, the O6 atom can act as the nucleophile in the second reaction step, leading to the extension of an α-glucan at its nonreducing end by one glucose residue.…”

“…S1A; 7,[11][12][13][14][15]. In the first step, the αð1 → 2Þ-glycosidic bond of sucrose is cleaved, fructose is released, and a glucosylenzyme intermediate is formed in which the glucosyl unit is covalently attached to the catalytic nucleophile via a β-glycosidic linkage.…”

Flexibility of truncated and full‐length glucansucrase GTF180 enzymes from Lactobacillus reuteri 180

“…Natural and modified aglucans also hold great potential in biotechnology, as well as in food and health-related applications, particularly because of their potential prebiotic properties [4]. To further tailor glucansucrases for such applications, it is crucial to understand how glucansucrases synthesize their products, and to comprehend the principles that determine their product specificities [4,7].…”

Search for a dextransucrase minimal motif involved in dextran binding

“…More recently, Shah et al [10] revealed the crucial role of the aromatic residues found in the YG repeats, which were suggested to ''stack'' with the sugar units of dextran or mutan. Finally, Moulis et al [11] showed that YG repeats are involved in the DSR-S polymerization reaction, in which they are proposed to act as anchoring zones for the growing dextran chains during their elongation.…”