Investigations on the Determinants Responsible for Low Molar Mass Dextran Formation by DSR-M Dextransucrase

Abstract

Certain enzymes of the GH70 family dextransucrases synthesize very high molar mass dextran polymers, whereas others produce a mixed population of very high and low molar mass products directly from sucrose substrate. Identifying the determinants dictating polymer elongation would allow the tight control of dextran size. To explore this central question, we focus on the recently discovered DSR-M enzyme from Leuconostoc citreum NRRL B-1299, which is the sole enzyme that naturally, exclusively, and very efficiently produces only low molar mass dextrans from sucrose. Extensive biochemical and structural characterization of a truncated form of DSR-M (DSR-MΔ2, displaying the same biochemical behavior as the parental enzyme) and X-ray structural analysis of complexes with sucrose and isomaltotetraose molecules together with accurate monitoring of the resulting polymer formation reveal that DSR-MΔ2 adopts a nonprocessive mechanism attributed to (i) a high propensity to recognize sucrose as a preferred acceptor at the initial stage of catalysis, (ii) an ability to elongate oligodextrans irrespective of their size, and (iii) the presence of a domain V showing a weak ability to bind to the growing dextran chains. In this study, we present the 3D structure with the largest defined domain V reported to date in the GH70 family and map sugar binding pockets on the basis of the structure of the complex obtained with isomaltotetraose. Altogether, these findings give insights into the interplay between the domain V and the catalytic site during polymerization. They open promising strategies for GH70 enzyme engineering aiming at modulating glucan size.

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V

“…However, less is known on the enzyme structural determinants controlling the polymer size. To date, five 3D-structures of recombinant and truncated glucansucrases have been solved: the dextransucrase GTF180-ΔN from Lactobacillus reuteri 180 [12] that produces a dextran containing around 30% of α-1,3 osidic linkages and 60% of α-1,6 osidic ones, the mutansucrase GTF-SI from Streptococcus mutans [13], the reuteransucrase GTFA-ΔN from L. reuteri 121 [14], the dextransucrase DSR-M∆2 from L. citreum NRRL B-1299 [15] producing a linear α-1,6 dextran, and the alternansucrase ASR∆2 from L. citreum NRRL B-1355 [16]. All these GSs have in common a 5-domain organization with a global U-shape folding.…”

“…Domains A, B and C are also found in related GH13 enzymes, whereas domains IV and V are unique to the GH70 family. Among all the characterized glucansucrases, DSR-M from L. citreum NRRL B1299 is rather unusual as it is the first dextransucrase shown to produce only Low Molar Mass (LMM) dextrans of around 20-30 kg/mol directly from sucrose, and quasi-strictly composed α-1,6 osidic bonds [15]. The recent structural analysis of DSR-M∆2 in complex with isomaltooligosaccharides, completed by biochemical characterization and mutagenesis studies, allowed us to propose a scenario explaining the distributive mode of LMM dextran synthesis by this enzyme.…”

Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract

“…It also remains to be studied whether the repertoire of a-glucans synthesized by the starch-converting GtfB, GtfC and GtfD GH70 enzymes could be further expanded by protein engineering approaches. Very recently, the structure of the DSR-M dextransucrase from Leuconostoc citreum NRRL B-1299, producing a single low molar mass dextran has been reported providing important clues about the structural features determining glucan size (Claverie et al 2017).…”

Enzymatic synthesis of polysaccharide-based copolymers

“…28 The dextransucrase from Leuconostoc citreum NRRL B-1299 (DSR-M Δ1) was produced as a (His) 6 -DSR-M Δ1 fusion protein by the recombinant E. coli BL21 star strain carrying the pET-53-DESTdsrMΔ1 plasmid. 27 (His) 6 -DgAS, (His) 6 -ASR and (His) 6 -DSR-M Δ1 were further purified by means of affinity chromatography using ProBond nickel-charged resin (Invitrogen), following the manufacturer's instructions, as described elsewhere. 27,42 In order to make the reading of this study easier, (His) 6 -DgAS, (His) 6 -ASR and (His) 6 -DSR-M Δ1 will be referred to hereafter as DgAS, ASR and DSR-M respectively.…”

“…Unlike other dextransucrases, which usually produce very high molar mass polymers, this enzyme produces only low molar mass linear α(1 → 6)-linked dextran. 26,27 Alternansucrase (ASR; E.C. 2.4.1.140; CAZy family GH70) specificity is also remarkable due to the mixture of α(1 → 6) and α(1 → 3) linkages found in the main polymer chain.…”