6VHQ

Crystal structure of Bacillus subtilis levansucrase (D86A/E342A) in complex with oligosaccharides

Summary for 6VHQ

• Entry DOI: 10.2210/pdb6vhq/pdb
• Related: 1oyg 2vdt
• Related PRD ID: PRD_900051
• Descriptor: Glycoside hydrolase family 68 protein, beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose-(2-6)-beta-D-fructofuranose, beta-D-fructofuranose-(2-6)-beta-D-fructofuranose, ... (6 entities in total)
• Functional Keywords: levansucrase, transferase, glycoside hydrolase, levan, fructose polymers
• Biological source: Bacillus subtilis
• Total number of polymer chains: 2
• Total formula weight: 108835.76

Authors

Diaz-Vilchis, A.,Raga-Carbajal, E.,Rojas-Trejo, S.,Olvera, C.,Rudino-Pinera, E. (deposition date: 2020-01-10, release date: 2021-01-13, Last modification date: 2023-10-11)

Primary citation

Raga-Carbajal, E.,Diaz-Vilchis, A.,Rojas-Trejo, S.P.,Rudino-Pinera, E.,Olvera, C.
The molecular basis of the nonprocessive elongation mechanism in levansucrases.
J.Biol.Chem., 296:100178-100178, 2020

PubMed 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.

PubMed: 33303628
DOI: 10.1074/jbc.RA120.015853

Experimental method

X-RAY DIFFRACTION (2.047 Å)