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.

项目概要 自由基SAM（RS）酶在生物化学、微生物学、癌症、 这些酶的特点是其 [4Fe-4S] 簇有助于还原。 SAM 裂解为甲硫氨酸和 5'-脱氧腺苷基自由基 (5'-dA•)，后者能够提取 在过去的十年中，从各种可能的底物中产生氢（H•），提供了奇异的生物反应。 许多研究都集中在基于钴胺素 (Cbl) 的 RS 酶亚类上。 生物信息学中，RS 超家族的这个亚类目前包含超过 50,000 种可能的酶， 其中绝大多数是未注释的，此类中的大多数注释酶都是甲基化酶。 然而，也有非自由基和非甲基化酶的例子。 此类酶的反应性使 Cbl 在反应中的作用进一步复杂化 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，不会影响 BchE 的作用。 相反，它催化底物 Mg-原卟啉-的两次氧化和闭环。 IX 单甲酯。该反应在细菌叶绿素 a 的生物合成过程中形成第五环。 Cbl 依赖性 RS 酶之间的独特反应，该酶的体外研究由于以下原因而受到阻碍 其众所周知的不溶性，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。 它能够将底物转化为新物种，尽管不是该提案所概述的预测的最终产品。 BchE 的光谱和电化学表征作为每个辅因子（钴胺素和 Fe- 此外，实现最终产品完全反应的实验是 这些包括确定哪些蛋白质结合伴侣和天然还原剂是必要的。 最后，还提出了诱导完全反应的替代策略。 BchE 是通过 X 射线晶体学提出的，人们对确定其结构非常感兴趣。 特征影响钴胺素辅因子在这种独特的反应中进行，这与更传统的反应相反 Cbl 依赖性 RS 酶中观察到的甲基化酶化学。