Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase MeMan5A and Their Role in Hydrolysis and Transglycosylation

被引：3

作者：

Birgersson, Simon ^{[1
]}

Morrill, Johan ^{[1
]}

Stenstroem, Olof ^{[2
]}

Wiemann, Mathias ^{[1
]}

Weininger, Ulrich ^{[2
,3
]}

Soederhjelm, Paer ^{[2
]}

Akke, Mikael ^{[2
]}

Stalbrand, Henrik ^{[1
]}

机构：

[1] Lund Univ, Dept Chem, Biochem & Struct Biol, POB 124, S-22100 Lund, Sweden

[2] Lund Univ, Dept Chem, Biophys Chem, POB 124, S-22100 Lund, Sweden

[3] Martin Luther Univ Halle Wittenberg, Inst Phys Biophys, D-06108 Halle, Germany

来源：

CATALYSTS | 2023年 / 13卷 / 09期

基金：

瑞典研究理事会;

关键词：

beta-mannanase; enzymatic synthesis; transglycosylation; flexibility; novel glycosides; MYTILUS-EDULIS; ENZYMATIC CHARACTERIZATION; BACKBONE DYNAMICS; HYDROLASE; LOOP; ENDO-BETA-1,4-MANNANASES; PURIFICATION; EXPRESSION; SOFTWARE; SEQUENCE;

D O I：

10.3390/catal13091281

中图分类号：

O64 [物理化学（理论化学）、化学物理学];

学科分类号：

070304 ; 081704 ;

摘要：

beta-Mannanases hydrolyze beta-mannans, important components of plant and microalgae cell walls. Retaining beta-mannanases can also catalyze transglycosylation, forming new beta-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (Mytilus edulis) GH5_10 beta-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme's catalytic efficiency (k(cat)/K-m using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by O-18 labeling of hydrolysis products and mass spectrometry. Results show that the substitution of both tryptophans was required to shift away from the dominant binding mode of mannopentaose (spanning subsites -3 to +2), suggesting that both tryptophans contribute to glycan binding. NMR spectroscopy and molecular dynamics simulations were conducted to analyze protein flexibility and glycan binding. We suggest that W240 is rigid and contributes to +2 subsite mannosyl specificity, while W281 is flexible, which enables stacking interactions in the +2 subsite by loop movement to facilitate binding. The substitutions significantly reduced or eliminated transglycosylation with saccharides as glycosyl acceptors but had no significant effect on reactions with alcohols.

引用

页数：20