Viegas Matilde F, Neves Rui P P, Ramos Maria J, Fernandes Pedro A
Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal.
Chemphyschem. 2022 Oct 19;23(20):e202200269. doi: 10.1002/cphc.202200269. Epub 2022 Aug 4.
Hydrolysis of lignocellulosic biomass, composed of a lignin-carbohydrate-complex (LCC) matrix, is critical for producing bioethanol from glucose. However, current methods for LCC processing require costly and polluting processes. The fungal Thermothelomyces thermophila glucuronoyl esterase (TtGE) is a promising thermophilic enzyme that hydrolyses LCC ester bonds. This study describes the TtGE catalytic mechanism using QM/MM methods. Two nearly-degenerate rate-determining transition states were found, with barriers of 16 and 17 kcal ⋅ mol , both with a zwitterionic nature that results from a proton interplay from His346 to either the Ser213-hydroxyl or the lignin leaving group and the rehybridisation of the ester moiety of the substrate to an alkoxide. An oxyanion hole, characteristic of esterases, was provided by the conserved Arg214 through its backbone and sidechain. Our work further suggests that a mutation of Glu267 to a non-negative residue will decrease the energetic barrier in ca. -5 kcal ⋅ mol , improving the catalytic rate of TtGE.
由木质素 - 碳水化合物复合物（LCC）基质组成的木质纤维素生物质的水解对于从葡萄糖生产生物乙醇至关重要。然而，目前处理LCC的方法需要成本高昂且有污染的过程。嗜热栖热菌葡糖醛酸酰基酯酶（TtGE）是一种很有前景的嗜热酶，可水解LCC酯键。本研究使用量子力学/分子力学（QM/MM）方法描述了TtGE的催化机制。发现了两个近乎简并的速率决定过渡态，其势垒分别为16和17 kcal·mol⁻¹，两者都具有两性离子性质，这是由His346向Ser213 - 羟基或木质素离去基团的质子相互作用以及底物酯部分重新杂化形成醇盐导致的。保守的Arg214通过其主链和侧链提供了酯酶特有的氧负离子洞。我们的工作进一步表明，将Glu267突变为非负残基将使能量势垒降低约 -5 kcal·mol⁻¹，提高TtGE的催化速率。
