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 Thermobifida fusca 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 A₆B₆E₆ stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A₃ and a B₆E₆ 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 Streptomyces coelicolor, 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.

酰基辅酶A羧化酶（AcCCase）是生物素依赖性酶，能够将一个以上的短链酰基辅酶A底物羧化。我们已经对这种AcCCase进行了结构和动力学分析，嗜热栖热菌YX，在羧化乙酰辅酶A，丙酰辅酶A和丁酰辅酶A时表现出混杂性。该酶由两个催化亚基（TfAcCCA和TfAcCCB）和一个非催化亚基TfAcCCE组成，并以A ₆ B ₆ E ₆化学计量的四级结构组织。而且，该全酶结构似乎主要是由两个A ₃和B ₆ E ₆亚复合物组装而成。TfAcCCE亚基的作用是促进全酶复合物的组装，从而激活催化作用。基于先前对AcCCase的研究天蓝色链霉菌，我们探讨了TfAcCCB亚基中保守的Asp残基是否可能在确定这些类型酶的底物选择性中起作用。突变此D427残基会导致TfAcCCase的底物特异性发生变化，从而提高羧化乙酰辅酶A的能力，同时降低丙酰辅酶A和丁酰辅酶A的羧化能力。这些结果共同表明，AcCCB亚基的D427残基是重要的，但不是AcCCase酶底物特异性的唯一决定因素。