Discovery, Structure, and Function of Filamentous 3-Methylcrotonyl-CoA Carboxylase

Abstract

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments and determined its structure at near-atomic resolution using cryoEM. α6β6 LtMCC dodecamers assemble in a twisted-stacks architecture, manifesting as supramolecular rods extending up to approximately 400 nanometers. LtMCCs in filaments bind biotin but are not covalently biotinylated and lack coenzyme A. Filaments elongate by stacking α6β6 LtMCCs onto the exterior α-trimer of the terminal α6β6 dodecamer. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state within the mitochondrial matrix. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the functional significance of polymerization in the carboxylase superfamily and beyond.

Filament structures unveil the dynamic organization of human acetyl-CoA carboxylase

“…An exo-site (or "exo pocket") for biotin was first identified in Staphylococcus aureus PYC (20). Biotin was also found to bind at the pocket far away from the catalytic sites in the CT domains of ACC, PCC and MCC, indicating the conservation of the exo-site in BDC family (8,21,22). According to the well-accepted two-step reaction mechanism for the carboxylation of acetyl-CoA, the covalently linked biotin is first carboxylated in the BC domain and then translocated to the CT domain to transfer the carboxyl group to acetyl-CoA (1,6,23).…”