Loose Jennifer S M, Arntzen Magnus Ø, Bissaro Bastien, Ludwig Roland, Eijsink Vincent G H, Vaaje-Kolstad Gustav
Faculty of Chemistry, Biotechnology and Food Science , Norwegian University of Life Sciences (NMBU) , 1432 Ås , Norway.
BOKU-University of Natural Resources and Life Sciences , Department of Food Science and Technology, Biocatalysis and Biosensing Laboratory , 1180 Vienna , Austria.
Biochemistry. 2018 Jul 17;57(28):4114-4124. doi: 10.1021/acs.biochem.8b00484. Epub 2018 Jun 29.
Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the degradation of polysaccharides in biomass by catalyzing powerful oxidative chemistry using only a single copper ion as a cofactor. Despite the natural abundance and importance of these powerful monocopper enzymes, the structural determinants of their functionality have remained largely unknown. We have used site-directed mutagenesis to probe the roles of 13 conserved amino acids located on the flat substrate-binding surface of CBP21, a chitin-active family AA10 LPMO from Serratia marcescens, also known as SmLPMO10A. Single mutations of residues that do not interact with the catalytic copper site, but rather are involved in substrate binding had remarkably strong effects on overall enzyme performance. Analysis of product formation over time showed that these mutations primarily affected enzyme stability. Investigation of protein integrity using proteomics technologies showed that loss of activity was caused by oxidation of essential residues in the enzyme active site. For most enzyme variants, reduced enzyme stability correlated with a reduced level of binding to chitin, suggesting that adhesion to the substrate prevents oxidative off-pathway processes that lead to enzyme inactivation. Thus, the extended and highly evolvable surfaces of LPMOs are tailored for precise multipoint substrate binding, which provides the confinement that is needed to harness and control the remarkable oxidative power of these enzymes. These findings are important for the optimized industrial use of LPMOs as well as the design of LPMO-inspired catalysts.
裂解多糖单加氧酶（LPMOs）通过仅使用单个铜离子作为辅因子催化强大的氧化化学反应，在生物质中多糖的降解过程中发挥着关键作用。尽管这些强大的单铜酶在自然界中广泛存在且很重要，但其功能的结构决定因素在很大程度上仍不清楚。我们利用定点诱变技术，探究了位于粘质沙雷氏菌的几丁质活性AA10家族LPMO（也称为SmLPMO10A）CBP21平坦底物结合表面上的13个保守氨基酸的作用。与催化铜位点不相互作用、而是参与底物结合的残基单点突变，对酶的整体性能产生了显著的强烈影响。对产物随时间形成的分析表明，这些突变主要影响酶的稳定性。使用蛋白质组学技术对蛋白质完整性进行研究表明，活性丧失是由酶活性位点中必需残基的氧化引起的。对于大多数酶变体而言，酶稳定性降低与几丁质结合水平降低相关，这表明与底物的粘附可防止导致酶失活的氧化旁路过程。因此，LPMOs扩展且高度可进化的表面专为精确的多点底物结合而定制，这种结合提供了利用和控制这些酶强大氧化能力所需的限制条件。这些发现对于LPMOs的优化工业应用以及受LPMO启发的催化剂设计具有重要意义。
