Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Engineering, China Agricultural University, Beijing 100083, China.
School of Biotechnology, State Key Laboratory of Bioreactor Engineering, R&D Center of Separation and Extraction Technology in Fermentation Industry, East China University of Science and Technology, Shanghai 200237, China.
Biochim Biophys Acta Gen Subj. 2018 Jun;1862(6):1376-1388. doi: 10.1016/j.bbagen.2018.03.016. Epub 2018 Mar 14.
Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. β-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-β-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pH 5.0 and 50 °C, and exhibited high specific activity towards locust bean gum (2337 U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A-M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (-3 to +4) in the catalytic groove of RmMan134A. Mannose in the -1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impedes to bond with galactose branches. In addition, the various binding modes of RmMan134A-M5 indicated that subsites -2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A-M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and optimal conditions of RmMan134A were significantly improved. The purpose of this paper is to investigate the structure and function of fungal GH family 134 β-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.
甘露聚糖是半纤维素的主要组成部分之一，它是一种来自高等植物的可再生资源。β-甘露聚糖酶是能够降解木质纤维素生物质的酶。在这里，我们克隆并表达了来自粗糙脉孢菌（Rhizopus microsporus）的内切-β-甘露聚糖酶（RmMan134A）。重组 RmMan134A 在 pH 5.0 和 50°C 时表现出最大活性，并且对罗望子胶具有高比活性（2337 U/mg）。为了深入了解 RmMan134A 的底物结合机制，我们进一步解析了四个复合物结构（RmMan134A-M3、RmMan134A-M4、RmMan134A-M5 和 RmMan134A-M6）。这些结构表明，在 RmMan134A 的催化槽中至少存在七个亚位点（-3 到+4）。-1 亚位点的甘露糖与 His113 和 Tyr131 形成氢键，呈现独特的构象。Lys48 和 Val159 形成空间位阻，阻碍与半乳糖支链结合。此外，RmMan134A-M5 的各种结合模式表明，在水解过程中-2 到+2 亚位点是不可或缺的。RmMan134A-M4 的结构表明，只有甘露糖六糖结合在+1 到+4 亚位点，因此 RmMan134A 不能水解聚合度≤4 的甘露聚糖寡糖。通过合理设计，显著提高了 RmMan134A 的比活性和最适条件。本文旨在研究真菌 GH 家族 134 β-1,4-甘露聚糖酶的结构和功能，以及 GH 家族 134 成员的底物结合机制。
