Structural investigations of adenosylcobalamin-dependent enzyme maturation

Abstract

Metalloenzymes utilize metallocofactors, ranging from single metal ions to complicated metallic clusters, to catalyze a wide range of challenging chemical reactions that are critical for life. Incorporation of these metallocofactors often relies on proteins known as metallochaperones that transport, modify, and/or insert the metallocofactor for its target metalloenzyme. This thesis focuses on the maturation of methylmalonyl-CoA mutase (MCM). In humans, MCM is the only known adenosylcobalamin (AdoCbl)- dependent enzyme; mutations or deletions of MCM or any metallochaperones involved in its maturation lead to methylmalonic aciduria, an inborn error of metabolism. The final step of MCM’s maturation involves an adenosyltransferase (ATR), which catalyzes the adenylation reaction to form AdoCbl and then delivers AdoCbl to MCM, and a G-protein chaperone, which facilitates AdoCbl delivery by the ATR through GTP hydrolysis. In addition to the human system, there are two bacterial systems used to understand the maturation of MCM: a homologous three-component system from Methylobacterium extorquens and an analogous two-component system from Cupriavidus metallidurans. The two-component system consists of the natural fusion protein, IcmF, which contains the AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, and the analogous ATR. This thesis provides structural and biochemical characterizations of these two model systems to understand how the metallochaperones, specifically the G-protein chaperones, enable efficient mutase maturation. We present the crystal structure of a minimal system consisting of the G-protein chaperone, MeaB, and the Cbl-binding domain of the MCM from M. extorquens. This structure trapped an active conformation of the G-protein chaperone, revealing the first snapshots of the 180° rotation of one protomer needed to complete the nucleotide binding site and perform GTP hydrolysis. We also present mutagenesis and solution state data for IcmF from C. metallidurans that characterize its nucleotide- and cofactor-state dependent oligomerization, important for cofactor loading and unloading. Using cryogenic electron microscopy, we obtain structural data on IcmF that show that the monomeric G-protein domains of IcmF dimerize to resemble the active conformation of dimeric MeaB, and that this “active” conformation of the G-protein domains physically props open the mutase domains to enable AdoCbl loading. Finally, we present the crystal structure of C. metallidurans ATR and use this structure in computational docking studies with C. metallidurans IcmF to probe the potential interfaces within a G-protein:ATR:mutase complex. Overall, this work deepens our understanding of the function of G-protein chaperones in the maturation of AdoCbl-dependent mutases and sets the stage for further studies of metallochaperones and their roles in metalloenzyme maturation. Collectively, these studies have implications in both human health and in biotechnology.