Mechanistic and spectroscopic investigations of pyruvate formate-lyase activating enzyme
Hutcheson, Rachel Ann Udelhoven.
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Radical S-adenosylmethionine (SAM) enzymes are a large and rapidly growing superfamily composed of thousands of members catalyzing a wide diversity of reactions by utilizing a reduced [4Fe-4S] ¹ + cluster and SAM to create a 5'-deoxyadenosyl radical capable of initiating controlled radical chemistry in important and difficult biochemical reactions. The prevalence of radical SAM enzymes in all kingdoms of life underscores the central role played by these enzymes. For the vast majority of putative radical SAM enzymes little is known regarding the reaction catalyzed or the mechanism of catalysis. Nevertheless, it is possible to gain insight into these enzymes from the radical SAM enzyme pyruvate formate-lyase activating enzyme (PFL-AE), which catalyzes the formation of a catalytically essential glycyl (G734) radical of pyruvate formate-lyase (PFL). The studies presented herein provide further understanding and characterization of PFL-AE as well as other radical SAM enzymes. The relevance and effect of the monovalent cation found in the active site of PFL-AE upon further analysis of the crystal structure was probed using coupled enzyme activity assays. Five different monovalent cations, Na +, K +, NH 4 +, Rb +, and Cs +, were investigated by calculating the specific activity of PFL-AE in the presence of each. PFL-AE was active in the presence of all tested cations, with specific activities correlating with cation size. Nuclear resonance vibrational spectroscopy performed on PFL-AE with an ⁵⁷ Fe labeled cluster showed a enzyme stiffening around the cluster and elongation of Fe-S bonds upon substrate and substrate analog binding. Rapid freeze-quench was used to mix PFL-AE with PFL and SAM on a millisecond time scale. The resulting samples were analyzed by electron paramagnetic resonance, which revealed a newly observed radical intermediate. To attempt characterization of this radical intermediate, electron nuclear double resonance spectroscopy (ENDOR) was used with site-specifically labeled SAM. The ENDOR signal detected was too weak to be analyzed; however, other labeled SAM molecules will be used in the future. To help further expand knowledge of radical SAM enzymes, an initial characterization of a putative methylthiotransferase (a subclass of the radical SAM superfamily) was undertaken. Results indicated that the enzyme methylthiolated a ribosomal small protein and not tRNA.