Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase
Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase
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DOI：
10.1111/febs.14782
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发表时间：
2019-05-01
期刊：
影响因子：
5.4
通讯作者：
Nonato, Maria Cristina
中科院分区：
文献类型：
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作者：
Ajalla Aleixo, Mariana A.;Rangel, Victor L.;Nonato, Maria Cristina
富马酸水合酶（FH，脱氢酶）催化富马酸可逆转化为l-苹果酸。FH分布于所有生物体中，在能量产生、DNA修复和肿瘤抑制中起重要作用。它们是人类代谢紊乱研究中非常重要的靶点，也是被忽视的热带疾病和结核病的潜在治疗靶点。本研究采用酶动力学、差示扫描荧光法和X射线晶体学方法对人FH进行了表征。对于第一次，两个基板的贡献进行了分析，同时在一个单一的动力学测定允许量化的动力学可逆反应的贡献。该蛋白的晶体结构属于空间群C222（1），晶胞参数a=125.43，B=148.01，c=129.76。通过分子置换解析结构，并以1.8埃分辨率进行精制。在我们的研究中，发现HEPES分子通过一系列包括Lys 467在内的相互作用，在C末端结构域（结构域3）与HsFH相互作用，之前描述为参与变构调节。当在HEPES存在下时，HsFH催化效率更高。残基467处的突变已经与FH缺陷引起的遗传疾病有关，这表明HEPES结合位点可能对酶动力学很重要。这项研究有助于理解HsFH结构及其与突变、酶缺乏和病理学的关系。
Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C222(1), with unit-cell parameters a=125.43, b=148.01, c=129.76. The structure was solved by molecular replacement and refined at 1.8 angstrom resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.