Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From YX.

Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a ABE stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A and a BE subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from , we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.