The molecular basis of the nonprocessive elongation mechanism in levansucrases.

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 (FOS) 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 wild type (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Most importantly, compared to the WT, the variants progressively yielded smaller-sized low-molecular weight (LMW) levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular weight (HMW) levans. Furthermore, an additional oligosaccharide-binding (OB) site 20 A 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.