Structural Basis of Substrate Selectivity and Catalysis in the Mycobacterial Long-Chain Acyl-CoA Carboxylase

Long-chain acyl-CoA carboxylase (LCC) is an essential enzyme complex in mycobacteria that generates acyl-CoA precursors for mycolic acid and complex lipid biosynthesis, yet its architecture and mechanism of substrate selection have remained unclear. Here we determine pre- and post-reaction states of the endogenous 868-kDa LCC complex from Mycobacterium smegmatis by cryo-electron microscopy at 2.1-3.7 [A] resolution. These structures visualize ATP-dependent redistribution of the biotin carboxyl carrier protein. LCC assembles into an asymmetric 8:2:4:2 organization of AccA3, AccD4, AccD5, and AccE5, with two biotin carboxylase modules flexibly tethered to a heterohexameric carboxyltransferase core. We define the structural basis of substrate selectivity within the CT core: AccD5 selectively binds the short-chain substrate C3-CoA, whereas AccD4 accommodates the long-chain substrate C16-CoA. In addition, we resolve AccD5-centered assemblies that associate with biotin carboxylase modules yet lack AccD4, providing structural evidence that distinct carboxyltransferase cores can engage shared modules to generate alternative holoenzyme architectures. Together, these findings define LCC and AccD5-centered assemblies as elements of a combinatorial acyl-CoA carboxylase platform and establish the structural principles governing assembly-specific function in mycobacteria.