Investigating the structural and mechanistic paramters of two radical SAM enzymes, spore photoproduct lyase and HydE
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Date
2013
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Montana State University - Bozeman, College of Letters & Science
Abstract
The resistance of Bacterial spores to UV radiation makes them a causative agent in many diseases and poses a threat to humans and animals alike. This unique resistance stems from the repair of a thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP)on exposure to UV irradiation. During the early stages of germination, this SP is repaired by an enzyme, spore photoproduct lyase (SPL) into two thymines. SPL is a member of the radical SAM superfamily of enzymes and requires S-adenosylmethionine (SAM) and a [4Fe-4S]1+/2+ cluster to perform its catalysis. The first part of this dissertation is dedicated towards understanding the solution phase dynamics of this protein on binding with its substrate and co-factor via hydrogen deuterium exchange. Analyses of the effects of SAM binding to SPL indicate that the protein does go through a conformational change localized around its active site. We have also demonstrated that the concomitant binding of SAM and dinucleotide SP contributes more significantly to the active site stabilization than what is observed with just SAM binding. Moreover we have provided initial evidence that the SPL might be utilizing the deformation of the phosphodiester back bone of SP to recognize, bind and initiate catalysis. We have unequivocally demonstrated that the catalytic [4Fe-4S] cluster plays a significant role in substrate/cofactor binding most likely due to the stabilization of the 8 residue loop region it resides on. The second part of this dissertation is focused towards understanding the role of maturase proteins in the assembly of the active site of [FeFe]- hydrogenase. The assembly and biosynthesis of the H-cluster requires three accessory enzymes HydE, HydG and HydF. Herein show that HydE utilizes cysteine as a substrate. We have also shown through LCMS and specifically deuterium labeled substrate, that catalysis is initiated via a H atom abstraction from the beta carbon of cysteine. Our investigations into the mechanism of HydG mediated turnover of tyrosine reveal that catalysis is initiated via a single H atom abstraction from the phenolic position of the substrate. Taken together we believe that our investigations have provided some critical insights into specific roles of these enzymes.