Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis
Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis
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基于底物辅助催化的蛋氨酸γ-裂合酶突变体降解同型半胱氨酸的结构和机制见解
DOI：
10.1002/pro.3158
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发表时间：
2017
期刊：
影响因子：
8
通讯作者：
Harada S.
中科院分区：
文献类型：
--
作者：
Sato D;Shiba T;Yunoto S;Furutani K;Fukumoto M;Kudou D;Tamura T;Inagaki K;Harada S.
甲硫氨酸γ-裂解酶（MGL）催化L-甲硫氨酸及其衍生物的α，γ-消除以及L-半胱氨酸及其衍生物的α，β-消除，以产生α-酮酸、挥发性硫醇和氨。MGL的反应机制已通过使用几种定点突变体的酶学研究进行了表征。恶臭假单胞菌MGL C116 H突变体对甲硫氨酸的降解活性显著降低，同时保留对同型半胱氨酸的活性。为了理解潜在的机制并辨别这些底物之间的细微差异，我们分析了反应中间体的晶体结构。C116 H突变体和甲硫氨酸之间形成的复合物表明，催化二聚体相邻亚基中的环结构（Ala 51-Asn 64）无法接近辅因子吡哆醛5′-磷酸（PLP），因为His 116破坏了Asp 241与Lys 240的相互作用，并且Lys 240的释放侧链导致该环的空间位阻。相反，在C116 H突变体和同型半胱氨酸之间形成的复合物中，与PLP缀合的底物的巯基部分通过水分子抵消His 116的咪唑环，破坏His 116和Asp 241的相互作用，并恢复Asp 241与Lys 240的相互作用。这些结构数据表明，Cys 116到His突变使酶对原始底物无活性，但当底物是同型半胱氨酸时，由于底物辅助催化，活性恢复。
Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination ofl‐methionine and its derivatives as well as the α, β‐elimination ofl‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. ThePseudomonas putidaMGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.