[Fe]-hydrogenase: biosynthesis of the FeGP cofactor and its catalytic function

[Fe]-氢化酶：FeGP辅因子的生物合成及其催化功能

• 批准号: 310432546
• 负责人: Dr. Seigo Shima, Ph.D.
• 金额: --
• 依托单位: Abteilung Biochemie und Synthetischer Metabolismus
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/310432546?language=en
• 关键词: Fe; hydrogenase; biosynthesis; FeGP; cofactor

The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of [Fe]-hydrogenase, which catalyzes reversible hydride transfer from H2 to methenyl-tetrahydromethanopterin. This reaction is involved in the hydrogenotrophic methanogenic pathway. In the FeGP cofactor, a low-spin Fe(II) is fixed by pyridinol-N and acyl-C from the pyridinol moiety, and the iron is coordinated with two CO ligands and one cysteine-S. Mutation analysis has indicated that the Cys176-S-Fe bonding is crucial for the activity of [Fe]-hydrogenase. This cofactor is biosynthesized by the reactions catalyzed by HcgA-G proteins. HcgA and HcgG are iron-sulfur cluster containing enzymes. We have previously identified the function of HcgB, HcgE and HcgF based on structural and biochemical analyses. We also proposed HcgD as an iron-trafficking protein for insertion of iron into the cofactor precursor. In the first half of this Priority program, we identified that the S-adenosyl-methionine-dependent methyltransferase-reaction of HcgC uses chemically synthesized pyridinol precursor and unveiled the unique catalytic mechanism of HcgC. Based on this finding, we established the methods for preparation of the guanylylpyridinol precursor from chemically synthesized pyridinol precursor using HcgB and HcgC. We also found a novel enzyme activity, which can repair the partially light-decomposed FeGP cofactor. In addition, we solved the crystal structure of [Fe]-hydrogenase in the closed active conformation at an atomic resolution (1.06 Å), in which the precise structure of the FeGP cofactor in the activated enzyme was indicated. Based on the active form structure, we proposed the catalytic functions of the FeGP cofactor. In the second half of this program, we would like to study further biosynthesis of the FeGP cofactor; we will use the in vitro biosynthesis methods using the chemically synthesized pyridinol precursor as the substrate. We would like to also characterize the enzyme activity of each Hcg protein using biochemical techniques and delta hcg mutants. We will study the enzyme activity that repairs the light-decomposed FeGP cofactor. As the second fundamental area expected for this Priority program, we will study the function of the FeGP cofactor in the catalytic mechanism of [Fe]-hydrogenase. For this aim, we will use several spectroscopic methods (infrared, Mössbauer, resonance Raman and Nuclear resonance vibrational spectroscopy) and density function theory calculation in collaboration with the groups in this Priority program.

铁-鸟苷基吡啶醇（FeGP）辅因子是[Fe]-氢化酶的辅基，其催化从H2到亚甲基-四氢甲蝶呤的可逆氢化物转移。该反应参与氢营养型产甲烷途径。在FeGP辅因子中，低自旋Fe（II）通过吡啶醇-N和来自吡啶醇部分的酰基-C固定，并且铁与两个CO配体和一个半胱氨酸-S配位。突变分析表明Cys 176-S-Fe键对[Fe]-氢化酶的活性至关重要。该辅因子通过HcgA-G蛋白催化的反应生物合成。HcgA和HcgG是含铁硫簇的酶。我们以前已经确定了HcgB，HcgE和HcgF的结构和生化分析的基础上的功能。我们还提出了HcgD作为铁运输蛋白插入到辅因子前体。在这个优先计划的前半部分，我们确定了HcgC的S-腺苷-甲硫氨酸依赖性甲基转移酶反应使用化学合成的吡啶醇前体，并揭示了HcgC独特的催化机制。基于这一发现，我们建立了使用HcgB和HcgC从化学合成的吡啶醇前体制备鸟苷基吡啶醇前体的方法。我们还发现了一种新的酶活性，可以修复部分光分解的FeGP辅因子。此外，我们解决了[Fe]-氢化酶的晶体结构在封闭的活性构象在原子分辨率（1.06 μ m），其中的FeGP辅因子在活化的酶的精确结构被指示。基于活性形式的结构，我们提出了FeGP辅因子的催化功能。在该计划的后半部分，我们希望进一步研究FeGP辅因子的生物合成;我们将使用体外生物合成方法，使用化学合成的吡啶醇前体作为底物。我们还希望使用生物化学技术和Δ hCG突变体来表征每个Hcg蛋白的酶活性。我们将研究修复光分解FeGP辅因子的酶活性。作为该优先计划的第二个基本领域，我们将研究FeGP辅因子在[Fe]-氢化酶催化机制中的功能。为此，我们将使用几种光谱方法（红外，穆斯堡尔，共振拉曼和核共振振动光谱）和密度函数理论计算与本优先计划中的小组合作。