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
基本信息
- 批准号:10751688
- 负责人:
- 金额:$ 6.95万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AnabolismAntibioticsBacteriochlorophyllsBindingBinding ProteinsBiochemistryBioinformaticsBiologicalCarbonCellsChemistryCobalaminCommunicable DiseasesCrystallizationDataElectron Spin Resonance SpectroscopyElectronsEnzymesFilmGoalsHydrogenIn VitroInfluentialsInvestigationMalignant NeoplasmsMethionineMethodsMethylationMethyltransferaseMicrobiologyMonitorMossbauer SpectroscopyNatural ProductsNatureOxidation-ReductionPlayPositioning AttributeProteinsReactionReducing AgentsRhodobacter capsulatusRoleS-AdenosylhomocysteineS-AdenosylmethionineStructureWorkX-Ray Crystallographycofactorcrosslinkdesignexperimental studyin vitro Assayinsightinterestmagnesium protoporphyrinoxidationprotoporphyrin IX
项目摘要
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.
项目总结
项目成果
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