Département de Biologie, Centre d'Étude et de Valorisation de la Diversité Microbienne, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
BMC Biochem. 2013 Sep 16;14:23. doi: 10.1186/1471-2091-14-23.
Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42.
Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity.
Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan.
链霉菌属 N174 壳聚糖酶（CsnN174）属于糖苷水解酶家族 46，是研究最多的壳聚糖酶之一。先前的研究确定了该反转酶的几个关键残基，例如两个催化羧酸氨基酸以及参与底物结合的残基。尽管在理解该壳聚糖酶的催化机制方面取得了进展，但 GH46 家族中高度保守的一些残基的功能尚未完全阐明。本研究集中于这样一个残基，精氨酸 42。
将 Arg42 突变为任何其他氨基酸都会导致酶活性急剧丧失。对 R42E 和 R42K 壳聚糖酶的详细研究表明，突变酶不仅在催化活性方面受到损害，而且在与底物的相互作用方式方面也受到损害。突变酶对底物抑制更为敏感，并且对各种脱乙酰度的壳聚糖的活性模式发生了改变。我们的数据表明，Arg42 在 CsnN174 活性中起双重作用。
精氨酸 42 对于维持壳聚糖酶 CsnN174 的酶学功能至关重要。我们建议，通过优化带负电荷的底物结合裂缝中的羧酸残基与壳聚糖中 GlcN 残基的氨基之间的静电相互作用，该精氨酸影响催化亲核残基和酶的底物结合模式。
