The molecular basis of the nonprocessive elongation mechanism in levansucrases

Abstract

Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (−1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity ( K m) values to the wildtype (WT) K m value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase

“…To avoid confusion, we term this the +1 acceptor (+1a) subsite followed by the +2a till +5a Fru subsites and terminating with the +6a Glc subsite (Figure 10). Intriguingly, this acceptor docking pose (Figure 10) follows a completely different direction than the one revealed for a levan-type oligosaccharide in a SacB D86A/E342A double mutant [91], suggesting that different acceptor binding paths are followed in these two different types of sucrases. Interestingly, our predicted inulin binding path perfectly overlapped with the position of a second Suc molecule in the inulosucrase structure of L. johnsonii (2YFS; [34]), but this requires further confirmation through mutagenesis and 3D-structure analysis of HmcIsc and an inulin oligosaccharide as acceptor.…”

Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity

“…Thus, it was proposed that the synthesis of levan is also affected by remote oligosaccharide binding sites in the LS structure . Indeed, in a recent study, the impact of remote levan-type FOS binding sites (OB1 and OB2) in Bs -SacB was observed with a cocrystallized complex of the enzyme (D86A/E342A mutant) with a levan-type FOS (6,6,6,6,6,6-kestooctaose) . Mutation of residues near +2, +3, and +4 subsites in Bs -SacB merely reduced the size of low molecular mass (LMW) levan (generally, 8 × 10 3 ≤ M W ≤ 5 × 10 4 Da), but not that of HMW levan.…”

“…8 Indeed, in a recent study, the impact of remote levan-type FOS binding sites (OB1 and OB2) in Bs-SacB was observed with a cocrystallized complex of the enzyme (D86A/E342A mutant) with a levan-type FOS (6,6,6,6,6,6-kestooctaose). 17 Mutation of residues near +2, +3, and +4 subsites in Bs-SacB merely reduced the size of low molecular mass (LMW) levan (generally, 8 × 10 3 ≤ M W ≤ 5 × 10 4 Da), but not that of HMW levan. In this regard, it was suggested earlier that the different product size specificities of G + and G − LS relate to processive and nonprocessive modes, respectively.…”

Structure–Function Relationship Studies of Multidomain Levansucrases from Leuconostocaceae Family

“…Inulosucrases (ISs) synthesize inulin, a polymer of fructose with β (2-1) bonds. The structural characteristics of these enzymes determine their regiospecificity and transference/hydrolysis specificity, as well as fructan size and branching [ 7 , 9 ].…”

“…The complete levan synthesis pathway has been recently identified, pinpointing the amino acids that make up the five product/acceptor binding subsites. These subsites help the enzyme carry out fructan elongation [ 9 ]. FNs can synthesize polymers with different molecular weights by means of two distinct elongation mechanisms.…”