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。这些酶的特征是它们的[4Fe-4S]簇促进还原 将SAM裂解为蛋氨酸和5‘-脱氧腺苷自由基(5’-da·)。后者能够抽象出一个 氢(H·)来自多种可能的底物，提供奇异的生物反应。在过去的十年里， 许多研究都集中在依赖钴胺(Cbl)的RS酶亚类上。基于 生物信息学，RS超家族的这个亚类目前有超过50,000种可能的酶， 其中绝大多数是未加注释的。这一类中的大多数带注释的酶是甲基酶，它可以 作用于未活化或惰性碳。然而，也有非自由基酶和非甲基酶的例子 来自这类酶的反应活性进一步复杂化了对Cb1在反应中所起作用的理解 机制。 这项拟议的工作旨在确定Cbl在BChE中的作用，BChE是一种依赖Cbl的RS酶，不 催化甲基转移。相反，它催化底物镁原卟啉的两次氧化和一次环合 单甲酯。这个反应在细菌叶绿素a的生物合成过程中形成了第五个环。虽然是一个 依赖Cbl的RS酶之间的独特反应，该酶的体外研究因以下原因而受阻 它臭名昭著的不解性。初步数据显示，布克实验室已经发现了这种酶的可溶结构 它能够将底物转化为新的物种，尽管不是预测的最终产品。这份提案概述了 研究了不同辅因子(钴胺和铁)对BChE的光谱和电化学性能的影响。 S集群)是不被理解的。此外，实现对最终产品的完全反应的实验是 概述。这些措施包括确定哪些蛋白质结合伙伴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，结构特征 用X-射线结晶学的方法提出了BCHE的性质。有很大的兴趣来确定什么结构 与更传统的反应相反，特征会影响钴胺辅助因子在这种独特的反应中进行 在依赖Cbl的RS酶中看到的甲基酶化学。