Elucidating the Role of Cobalamin in BchE, a B12-Binding Radical SAM Enzyme

阐明钴胺素在 BchE（一种 B12 结合自由基 SAM 酶）中的作用

• 批准号: 10751688
• 负责人: Nicholas James York
• 金额: $ 6.95万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-11-01 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751688
• 关键词: Anabolism; Antibiotics; Bacteriochlorophylls; Binding; Binding Proteins; Biochemistry; Bioinformatics; Biological; Carbon; Cells; Chemistry; Cobalamin; Communicable Diseases; Crystallization; Data; Electron Spin Resonance Spectroscopy; Electrons; Enzymes; Film; Goals; Hydrogen; In Vitro; Influentials; Investigation; Malignant Neoplasms; Methionine; Methods; Methylation; Methyltransferase; Microbiology; Monitor; Mossbauer Spectroscopy; Natural Products; Nature; Oxidation-Reduction; Play; Positioning Attribute; Proteins; Reaction; Reducing Agents; Rhodobacter capsulatus; Role; S-Adenosylhomocysteine; S-Adenosylmethionine; Structure; Work; X-Ray Crystallography; cofactor; crosslink; design; experimental study; in vitro Assay; insight; interest; magnesium protoporphyrin; oxidation; protoporphyrin IX

Project Summary Radical SAM (RS) enzymes have been greatly influential in the fields of biochemistry, microbiology, cancer, and infectious diseases. These enzymes are characterized by their [4Fe-4S] cluster which facilitates reductive cleavage of SAM to methionine and a 5’-deoxyadenosyl radical (5’-dA•). The latter of which is able to abstract a hydrogen (H•) from a wide range of possible substrates, affording exotic biological reactions. In the last decade, many studies have focused on the cobalamin (Cbl) dependent subclass of RS enzymes. Based on bioinformatics, this subclass of the RS superfamily currently accounts for over 50,000 possible enzymes, the vast majority of which are unannotated. Most of the annotated enzymes in this class are methylases which can act upon unactivated or inert carbons. However, there are examples of both non-radical and non-methylase reactivity from this class of enzymes which further complicate understanding the role Cbl plays in the reaction mechanism. This proposed work aims to determine the role of Cbl in BchE, a Cbl-dependent RS enzyme that does not catalyze methyl transfer. Instead, it catalyzes two oxidations and a ring closure of substrate, Mg-protoporphyrin- IX monomethylester. This reaction forms the fifth ring of bacteriochlorophyll a during its biosynthesis. Though a unique reaction among Cbl-dependent RS enzymes, in vitro studies of this enzyme have been impeded due to its notorious insolubility. Preliminary data shows the Booker lab has found a soluble construct of this enzyme which is able to convert substrate to a new species, though not the predicted final product. This proposal outlines the spectroscopic and electrochemical characterization of BchE as the role of each cofactor (cobalamin and Fe- S cluster) is not understood. Furthermore, experiments to fulfill complete reactivity to the final product are outlined. These include determining what protein binding partners and native reductants are necessary for the reaction. Alternative strategies for inducing full reactivity are also proposed. Finally, structural characterization of BchE is proposed by means of X-ray crystallography. There is much interest to determine what structural features influence the cobalamin cofactor to proceed in this unique reaction opposed to the more conventional methylase chemistry seen in Cbl-dependent RS enzymes.

项目摘要 自由基SAM（RS）酶在生物化学、微生物学、癌症、 和传染病。这些酶的特征在于它们的[4Fe-4S]簇， SAM裂解为甲硫氨酸和5 '-脱氧腺苷自由基（5'-dA·）。后者能够抽象出一个 氢（H·）从各种可能的底物，提供外来的生物反应。在过去十年中， 许多研究集中于RS酶的钴胺素（Cbl）依赖性亚类。基于 根据生物信息学，RS超家族的这个亚类目前占50，000多种可能的酶， 其中绝大多数都没有注释。这一类中的大多数注释的酶是甲基化酶，其可以 作用于未活化的或惰性的碳。然而，也有非自由基和非甲基化酶的例子 这类酶的反应性进一步使理解Cbl在反应中所起的作用复杂化 机制 这项拟议的工作旨在确定Cbl在BchE中的作用，BchE是一种Cbl依赖性RS酶， 催化甲基转移。相反，它催化底物Mg-原卟啉的两次氧化和一次闭环。 IX单甲酯。该反应在细菌叶绿素a的生物合成期间形成细菌叶绿素a的第五环。虽然 由于Cbl依赖性RS酶之间的独特反应，这种酶的体外研究由于以下原因而受到阻碍： 它臭名昭著的不溶性。初步数据显示布克实验室已经发现了这种酶的可溶性结构 其能够将底物转化为新的物质，尽管不是预测的最终产物。该提案概述了 BchE的光谱和电化学表征作为每个辅因子（钴胺素和Fe- S簇）不被理解。此外，实现对最终产物的完全反应性的实验是 概述。这些包括确定什么样的蛋白质结合伴侣和天然还原剂是必需的， 反应还提出了诱导完全反应性的替代策略。最后，结构表征 的BchE的提出通过X射线晶体学。有很多兴趣来确定什么结构 特征影响钴胺素辅因子在这种独特的反应中进行，而不是更常规的反应。 在Cbl依赖性RS酶中观察到的甲基化酶化学。