Chorozian Koar, Karnaouri Anthi, Georgaki-Kondyli Nefeli, Karantonis Antonis, Topakas Evangelos
Industrial Biotechnology & Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 15772, Athens, Greece.
Laboratory of General and Agricultural Microbiology, Department of Crop Science, Agricultural University of Athens, 11855, Athens, Greece.
Biotechnol Biofuels Bioprod. 2024 Feb 1;17(1):19. doi: 10.1186/s13068-024-02463-y.
The field of enzymology has been profoundly transformed by the discovery of lytic polysaccharide monooxygenases (LPMOs). LPMOs hold a unique role in the natural breakdown of recalcitrant polymers like cellulose and chitin. They are characterized by a "histidine brace" in their active site, known to operate via an O/HO mechanism and require an electron source for catalytic activity. Although significant research has been conducted in the field, the relationship between these enzymes, their electron donors, and HO production remains complex and multifaceted.
This study examines TthLPMO9G activity, focusing on its interactions with various electron donors, HO, and cellulose substrate interactions. Moreover, the introduction of catalase effectively eliminates HO interference, enabling an accurate evaluation of each donor's efficacy based on electron delivery to the LPMO active site. The introduction of catalase enhances TthLPMO9G's catalytic efficiency, leading to increased cellulose oxidation. The current study provides deeper insights into specific point mutations, illuminating the crucial role of the second coordination sphere histidine at position 140. Significantly, the H140A mutation not only impacted the enzyme's ability to oxidize cellulose, but also altered its interaction with HO. This change was manifested in the observed decrease in both oxidase and peroxidase activities. Furthermore, the S28A substitution, selected for potential engagement within the His1-electron donor-cellulose interaction triad, displayed electron donor-dependent alterations in cellulose product patterns.
The interaction of an LPMO with HO, electron donors, and cellulose substrate, alongside the impact of catalase, offers deep insights into the intricate interactions occurring at the molecular level within the enzyme. Through rational alterations and substitutions that affect both the first and second coordination spheres of the active site, this study illuminates the enzyme's function. These insights enhance our understanding of the enzyme's mechanisms, providing valuable guidance for future research and potential applications in enzymology and biochemistry.
溶菌多糖单加氧酶（LPMO）的发现深刻改变了酶学领域。LPMO在纤维素和几丁质等难降解聚合物的自然分解过程中发挥着独特作用。它们的活性位点具有“组氨酸支架”，已知通过O/HO机制发挥作用，且催化活性需要电子源。尽管该领域已开展了大量研究，但这些酶、它们的电子供体与HO产生之间的关系仍复杂且多面。
本研究考察了TthLPMO9G的活性，重点关注其与各种电子供体、HO以及纤维素底物的相互作用。此外，过氧化氢酶的引入有效消除了HO的干扰，从而能够基于电子传递至LPMO活性位点的情况准确评估每个供体的功效。过氧化氢酶的引入提高了TthLPMO9G的催化效率，导致纤维素氧化增加。当前研究对特定点突变有了更深入的了解，阐明了140位第二配位层组氨酸的关键作用。值得注意的是，H140A突变不仅影响了酶氧化纤维素的能力，还改变了其与HO的相互作用。这种变化表现为氧化酶和过氧化物酶活性均下降。此外，选择S28A替代是为了潜在参与His1 - 电子供体 - 纤维素相互作用三联体，其在纤维素产物模式上表现出依赖电子供体的变化。
LPMO与HO、电子供体和纤维素底物的相互作用，以及过氧化氢酶的影响，为深入了解酶内分子水平上发生的复杂相互作用提供了线索。通过影响活性位点第一和第二配位层的合理改变和替代，本研究阐明了酶的功能。这些见解增进了我们对酶机制的理解，为酶学和生物化学领域的未来研究及潜在应用提供了有价值的指导。
