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Development of an Oxygen-Tolerant Adenosyl Radical Enzyme for Directed-Evolution Applications

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Development of an Oxygen-Tolerant Adenosyl Radical Enzyme for Directed-Evolution Applications
Humble, Luke
The radical S-adenosyl-L-methionine (SAM) superfamily of enzymes (RSEs) is one of the most functionally diverse. Members of this group are involved in the biosynthesis of important natural products including vitamins, antivirals, and cofactors. These enzymes use a [4Fe–4S] cluster to deliver an electron to SAM, which, upon reduction, results in homolytic cleavage of the S─5’C bond, forming a 5’-deoxyadenosyl radical (5’-dAdo●). This radical then attacks the substrate—either by abstracting a substrate hydrogen atom or adducting to an olefinic functional group on the substrate. The [4Fe-4S] cluster, however, can be destroyed by oxygen, often requiring anaerobic conditions for any in vitro investigation into its function. This greatly hinders the ability to gain insights into the structural and kinetic characteristics that imbue the catalytic domain—a triose-phosphate isomerase (TIM) barrel—with its function. This limitation is one of the reasons why so few radical SAM enzymes have been biochemically characterized. We hypothesize that by replacing the O2-sensitive [Fe–S]:SAM-based 5’-dAdo●-trigger with one sourced from adenosylcobalamin (AdoCbl), we can still use the catalytic TIM barrel domain of the RSE to oversee conversion of the initial substrate radical to product. The results summarized in the subsequent chapters illustrate that such an approach is successful in rescuing thiamin production in a Δthic strain of E. coli. Specifically, AdoCbl-binding chaperone proteins MutS can interface with a truncated version of heterologously expressed ThiC (ThiCtrunc, missing the [4Fe–4S] cluster-binding domain) and still produce the native ThiC product, 4-amino-5-hydroxymethyl-2-methylpyrimidine-phosphate (HMP-P). Only when HMP-P is produced can Δthic E. coli survive on minimal media.
Glutamate Mutase
Oxygen Sensitivity
Radical SAM Enzymes
Stich, Troy A (advisor)
Dos Santos, Patricia C (committee member)
Donati, George L (committee member)
2023-01-24T09:35:34Z (accessioned)
2023-01-24T09:35:34Z (available)
2022 (issued)
Chemistry (discipline)
http://hdl.handle.net/10339/101763 (uri)
en (iso)
Wake Forest University

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