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 (Km) values to the wildtype (WT) Km 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.
Highlights
Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule
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
A low-molecular-weight (LMW, 7.2 kDa) levan is synthesized by employing a nonprocessive mechanism, in which the fructose units are added to growing chains but are taken up and released into the solution according to their affinity for the enzyme [10]
Summary
Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. A low-molecular-weight (LMW, 7.2 kDa) levan is synthesized by employing a nonprocessive mechanism, in which the fructose units are added to growing chains but are taken up and released into the solution according to their affinity for the enzyme [10]. These observations indicate that SacB has the structural machinery for both elongation mechanisms. In SacB, residues D86 and E342 have been identified as the nucleophile and the general acid/base, respectively, whereas D247 is the transition state stabilizer [8]
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