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首页> 外文期刊>Biochemistry >Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes
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Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes

机译：底物的多点精密结合保护裂解多糖单氧基酶免受自毁的途径过程

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摘要

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 SmLPM010A. 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.
机译：裂解多糖单氧化酶（LPMOS）在使用仅使用单铜离子作为辅助因子的铜离子的强大的氧化化学在生物质中的多糖降解中起着至关重要的作用。尽管这些强有力的单像酶的自然丰富和重要性，其功能的结构决定簇在很大程度上是未知的。我们已经使用定向诱变的诱变来探讨位于CBP21的扁平基材结合表面上的13个保守氨基酸的作用，从Serratia marcescens的丁蛋白活性家庭AA10 LPMO，也称为SMLPM010A。与催化铜部位不相互作用的残基的单一突变，而是参与底物结合对整体酶的性能具有显着的强烈影响。随着时间的推移分析产物形成表明，这些突变主要影响酶稳定性。使用蛋白质组学技术研究蛋白质完整性显示，酶活性位点中的必需残留物氧化引起的活性丧失。对于大多数酶变体，降低的酶稳定性与对甲壳素的结合水平降低相关，表明对基材的粘附性可防止导致酶失活的氧化脱途过程。因此，对于精确的多点衬底结合来定制LPMO的延伸和高度不可溶解的表面，这提供了利用并控制这些酶的显着氧化力所需的限制。这些发现对于LPMOS的优化工业用途以及LPMO启动催化剂的设计非常重要。

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  • 来源
    《Biochemistry》 |2018年第28期|共11页
  • 作者

    Loose Jennifer S. M.; Arntzen Magnus O.; Bissaro Bastien; Ludwig Roland; Eijsink Vincent G. H.; Vaaje-Kolstad Gustav;

  • 作者单位

    Norwegian Univ Life Sci NMBU Fac Chem Biotechnol &

    Food Sci As Norway;

    Norwegian Univ Life Sci NMBU Fac Chem Biotechnol &

    Food Sci As Norway;

    Norwegian Univ Life Sci NMBU Fac Chem Biotechnol &

    Food Sci As Norway;

    BOKU Univ Nat Resources &

    Life Sci Dept Food Sci &

    Technol Biocatalysis &

    Biosensing Lab A-1180 Vienna Austria;

    Norwegian Univ Life Sci NMBU Fac Chem Biotechnol &

    Food Sci As Norway;

    Norwegian Univ Life Sci NMBU Fac Chem Biotechnol &

    Food Sci As Norway;

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  • 正文语种 eng
  • 中图分类 生物化学;
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