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

Abstract

Abstract3-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 α- and β-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme fromTrypanosoma bruceiin its soluble, non-filamentous state at 2.5 Å resolution. We unambiguously observe the position of biotin, covalently-bound to the BBCP domain of α-subunits and occupying a novel binding pocket next to the active site of a neighboring β-subunit dimer. Moreover, flexibility of key residues at the α/α- and α/β-subunit interfaces enables pivoting of α-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.