Jitonnom Jitrayut, Sattayanon Chanchai, Kungwan Nawee, Hannongbua Supa
Division of Chemistry, School of Science, University of Phayao, Phayao 56000, Thailand.
Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
J Mol Graph Model. 2015 Mar;56:53-9. doi: 10.1016/j.jmgm.2014.12.002. Epub 2014 Dec 16.
Serratia marcescens chitinase B (SmChiB) catalyzes the hydrolysis of β-1,4-glycosidic bond, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. In this paper, the catalytic mechanism of SmChiB has been investigated by using density functional theory. The details of two consecutive steps (glycosylation and deglycosylation), the structures and energetics along the whole catalytic reaction, and the roles of solvent molecules as well as some conserved SmChiB residues (Asp142, Tyr214, Asp215, and Arg294) during catalysis are highlighted. Our calculations show that the formation of the oxazolinium cation intermediate in the glycosylation step was found to be a rate-determining step (with a barrier of 23 kcal/mol), in line with our previous computational studies (Jitonnom et al., 2011, 2014). The solvent water molecules have a significant effect on a catalytic efficiency in the degycosylation step: the catalytic water is essentially placed in a perfect position for nucleophic attack by hydrogen bond network, lowering the barrier height of this step from 11.3 kcal/mol to 2.9 kcal/mol when more water molecules were introduced. Upon the in silico mutations of the four conserved residues, their mutational effects on the relative stability of the reaction intermediates and the computed energetics can be obtained by comparing with the wild-type results. Mutations of Tyr214 to Phe or Ala have shown a profound effect on the relative stability of the oxazolinium intermediate, emphasizing a direct role of this residue in destabilizing the intermediate. In line with the experiment that the D142A mutation leads to almost complete loss of SmChiB activity, this mutation greatly decreases the stability of the intermediate, resulting in a very large increase in the activation barrier up to 50 kcal/mol. The salt-bridges residues (Asp215 and Arg294) were also found to play a role in stabilizing the oxazolinium intermediate.
粘质沙雷氏菌几丁质酶B（SmChiB）通过一种不同寻常的底物辅助机制催化β-1,4-糖苷键的水解，在该机制中底物自身充当分子内亲核试剂。本文利用密度泛函理论研究了SmChiB的催化机制。重点阐述了两个连续步骤（糖基化和去糖基化）的细节、整个催化反应过程中的结构和能量变化，以及溶剂分子和一些保守的SmChiB残基（Asp142、Tyr214、Asp215和Arg294）在催化过程中的作用。我们的计算表明，糖基化步骤中恶唑啉阳离子中间体的形成是速率决定步骤（势垒为23千卡/摩尔），这与我们之前的计算研究结果一致（Jitonnom等人，2011年、2014年）。溶剂水分子在去糖基化步骤中对催化效率有显著影响：催化水通过氢键网络基本上处于亲核攻击的理想位置，当引入更多水分子时，该步骤的势垒高度从11.3千卡/摩尔降至2.9千卡/摩尔。对四个保守残基进行计算机模拟突变后，通过与野生型结果比较，可以得到它们对反应中间体相对稳定性和计算能量的突变效应。将Tyr214突变为Phe或Ala对恶唑啉中间体的相对稳定性有深远影响，强调了该残基在使中间体不稳定方面的直接作用。与D142A突变导致SmChiB活性几乎完全丧失的实验一致，该突变极大地降低了中间体的稳定性，导致活化势垒大幅增加至50千卡/摩尔。还发现盐桥残基（Asp215和Arg294）在稳定恶唑啉中间体方面发挥作用。
