Structures of mycobacterial 3-methylcrotonyl-CoA carboxylase reveal carrier-domain translocation between catalytic sites.

3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes an essential step in leucine degradation. Here, we report two high-resolution cryo-EM structures of endogenous, biotin-bound Mycobacterium smegmatis AccA1-AccD1 MCC. The α6β6 complex adopts a canonical architecture with a hexameric carboxyltransferase core flanked by trimeric biotin carboxylase modules. The structures capture BCCP engaged at either the BC or CT active site, revealing long-range carrier-domain translocation. In the CT-engaged state, local BC-domain shifts disrupt the BC-site BCCP-interaction network, disfavoring BCCP rebinding. Unlike human MCC, no comparable CT-core remodeling is observed, suggesting distinct mechanisms of carrier-domain coordination.