How a mycobacterial enzyme builds the waxy barrier of TB bacteria

Mycobacteria, the family of bacteria that includes the tuberculosis-causing species, are wrapped in a dense, waxy cell wall built in part from mycolic acid. That unusual lipid layer makes the bacteria highly impermeable to many antibiotics and is therefore central to the pathogen’s resilience. Producing mycolic acid and related fatty acids such as tuberculostearic acid depends on building blocks made by a multi-part enzyme called the long-chain acyl-CoA carboxylase (LCC) complex. In work led by Yingke Liang, researchers set out to understand how this large assembly recognizes particular chemical substrates and how other proteins attach to it. The LCC complex contains multiple subunits with specific roles: AccA3, which carries both a biotin carboxylase (BC) domain and a biotin carboxyl carrier protein (BCCP) domain; AccD4 and AccD5, which are carboxyltransferases for long- and short-chain acyl-CoA; and AccE, a less well understood subunit. Using a mycobacterial model, Mycobacterium smegmatis, the team determined the structures of the intact LCC complex to reveal how its parts are arranged and how additional proteins can dock onto it.

The team used electron cryomicroscopy (cryo-EM) to capture the architecture of the LCC complex from Mycobacterium smegmatis, including snapshots taken during catalysis. The structures show a core heterohexamer made of AccD4 and AccD5 in a 2 AccD4:4 AccD5 arrangement, with eight AccA3 subunits linked to that core. Two AccE subunits act like tethers that attach the eight AccA3 subunits to the AccD4 2 AccD5 4 core. The work preserves important biochemical details: AccA3 carries the BC domain and the BCCP domain that bears biotin, and the LCC complex catalyzes carboxylation of the α-carbon of long-chain acyl-CoA as well as acetyl-CoA and propionyl-CoA. Cryo-EM images captured during activity reveal how substrate specificity is achieved: AccD5 binds tightly to CoA, favoring short-chain substrates, while AccD4 provides a binding site for long acyl chains. The BCCP domains of AccA3 move long distances to shuttle a carboxyl group from the BC domain of AccA3 to the acyl-CoA substrate bound in AccD5. In addition, the structures show that two copies of a protein complex made from MSMEG_0435 and MSMEG_0436 can bind the LCC complex and position the biotin moiety of BCCP domains near AccD5.

These structural snapshots clarify both the mechanical steps and the specificity rules that allow the LCC complex to make critical fatty-acid precursors for the mycobacterial cell wall. By showing how AccD4 and AccD5 differ in the way they recognize long acyl chains versus CoA, and how AccA3’s BCCP domains travel to deliver a carboxyl group, the work provides a detailed molecular picture of a central biosynthetic machine. The discovery that MSMEG_0435 and MSMEG_0436 form a complex that binds LCC and sequesters the biotin portion of BCCP domains near AccD5 points to a potential regulatory layer: the Mycobacterium tuberculosis ortholog Rv0263c is known to affect bacterial survival during transmission, so these interacting proteins may help control production of branched fatty acid precursors. Taken together, the structures give researchers a clear blueprint of how substrate choice and protein–protein interactions shape production of components that make the mycobacterial cell wall so impermeable.