The Molecular Basis of Improbable Enzymatic Chemisteries

Abstract

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 is a ubiquitous RNA modification, discovered at hundreds of sites in mRNAs. Pseudouridine synthases (Pus) are responsible for installing pseudouridine, 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.