Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC)

The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of α and β subunits which are encoded by distinct genes. Using immuno-affinity purification, an efficient protocol has been developed to purify the active MCCC which appears to reside in a ∼500-800 kDa complex in Superpose-6 gel-filtration chromatography. Consistent with the native enzyme, in the recombinant human MCCC, the stoichiometry of α and β subunits are at a one:one ratio. The kcat value of the recombinant enzyme is determined to be ∼4.0 s−1. It also possesses Km values (ATP: 45 ± 11 μM; 3-methylcrotonyl-CoA: 74 ± 7 μM) similar to those reported for the native enzyme. The recombinant human MCCC described here may provide a counter-screen enzyme source for testing cross reactivity for inhibitors against acetyl-CoA carboxylases which are designed to treat obesity, type 2 diabetes and other metabolic disorders.