6VHQ

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

6VHQ の概要

• エントリーDOI: 10.2210/pdb6vhq/pdb
• 関連するPDBエントリー: 1oyg 2vdt
• 関連するBIRD辞書のPRD_ID: PRD_900051
• 分子名称: 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)
• 機能のキーワード: levansucrase, transferase, glycoside hydrolase, levan, fructose polymers
• 由来する生物種: Bacillus subtilis
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 108835.76

構造登録者

Diaz-Vilchis, A.,Raga-Carbajal, E.,Rojas-Trejo, S.,Olvera, C.,Rudino-Pinera, E. (登録日: 2020-01-10, 公開日: 2021-01-13, 最終更新日: 2023-10-11)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.047 Å)