The cryo-EM structure of trypanosome 3-methylcrotonyl-CoAcarboxylase provides mechanistic and dynamic insights into enzymatic function

3-methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given its critical metabolic role, deficiencies in this enzyme associate with organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each - and {beta}-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in its soluble, non-filamentous state at 2.5 [A] resolution. We unambiguously observe the position of biotin, covalently-bound to the BBCP domain of -subunits and occupying a novel binding pocket next to the active site of a neighboring {beta}-subunit dimer. Moreover, flexibility of key residues at the /- and /{beta}-subunit interfaces enables pivoting of -subunit trimers to sequentially approach the otherwise distant active sites for the two steps in MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and assist drug discovery against trypanosome infections.