Dissertation

The Molecular Basis of Improbable Enzymatic Chemisteries

Purchal, Meredith K

2023

Overview

Enzymes are Nature’s best chemists’ and play a vital role in supporting the diverse chemistries fundamental to complex life. Given this central role, protein dysfunction can have serious biological implications. Proteins are defined by the Structure-Function relationship ubiquitous throughout nature, and these relationships can be exploited for detailed enzymatic characterization. Thus, I took an integrative structural and biochemical approach to establish the structural context and molecular basis of challenging chemistries catalyzed by three enzymes involved in mRNA modification and cobalamin-dependent processes, and whose dysfunction result in cancers, developmental- and metabolic disorders.Pseudouridine (Ψ) is a ubiquitous RNA modification, discovered at hundreds of sites in mRNAs. Pseudouridine synthases (Pus) are responsible for installing Ψ, but exactly how an individual Pus selects a specific target site is unclear. I sought to characterize the basis of Pus-RNA interactions in Pus7 and ultimately determined the contribution of substrate structure and conserved protein elements towards binding and catalysis. Pus7 is one of the predominant mRNA modifying Pus-enzymes, that exhibits distinct diversity in substrate selectivity, as well as increased activity under heat shock. I solved the structure of Saccharomyces cerevisiae Pus7 and visualized the architecture of the eukaryotic-specific insertions thought to contribute to expanded substrate scope. Indeed, the largest insertion (Insertion I) contains a nucleic acid binding R3H motif surrounded by positively charged residues. Subsequent analysis demonstrated that Insertion-I serves to fine-tune Pus7 activity in a substrate-dependent manner both in vitro and in cells. Further, this work revealed that Pus7 is extraordinarily promiscuous, modifying every substrate (both natural and non-natural) containing the consensus sequence without regard for structure. My work suggests that Pus7 selectivity is likely governed by additional factors including substrate accessibility and localization, rather than inherent enzyme properties.B12-dependent enzymes harness the unique organometallic properties of cobalt to catalyze a variety of challenging chemistries integral to single-carbon metabolism in all domains of life. In humans, there are two metabolically essential B12-dependent enzymes: methionine synthase (MS) and methylmalonyl-CoA mutase (MCM). Cobalamin-dependent MS is a multi-modular enzyme that employs remarkable molecular dynamics and domain rearrangements – deemed ‘molecular juggling’ – to catalyze three difficult methyl-transfer reactions at the site of the cobalt-cofactor. Biochemical challenges have hindered structural and mechanistic characterization of MS catalysis and conformational states. To address this, I describe a Thermus thermophilus MS variant that avoids the associated barriers of expression and purification. Using tMS as a model, I describe the first full-length structure of apoMS – finally visualizing all domains at once and gaining insights into the structural basis of B12-incorporation. Further, we captured MS with the Folate-domain oriented above the B12-domain, and cobalt is within the predicted distance for catalysis, and this likely represents the first catalytic structure captured for any corrinoid protein.MCM utilizes 5’-deoxyadenosylcobalamin (AdoCbl) to catalyze the interconversion of methylmalonyl-CoA to succinyl-CoA through homolysis of Co-C bond. Here, we determined the structure of Mycobacterium tuberculous MCM complexed with the suicide inactivator itaconyl-CoA, a succinyl-CoA analog. Notably, EPR studies confirm that we captured an air-stable biradical comprising a tertiary carbon radical (5’-deoxyadenosyl) coupled to the metal-centered cob(II)alamin radical in crystallo. Thus, in addition to describing the mechanism of I-CoA inhibition, these experiments provide molecular insights into how MCM controls radical trajectories during catalysis.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Biology / Biophysics
• ISBN: 9798379564377