Structural insight into mycobacterial cell wall biosynthesis by AftB, a potential anti-TB drug target.

Tuberculosis (TB) is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a significant health problem worldwide. The success of Mtb as the leading pathogen can largely be attributed to its atypical multilayered cell wall, which is highly impermeable and confers natural protection against attack from the immune system and many antibiotics. Due to its unique character, the mycobacterial cell wall has become the most widely exploited target of anti-TB drugs. AftB is an arabinofuranosyltransferase that functions in the terminal step of cell wall arabinan biosynthesis, catalyzing the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) onto the cell wall polysaccharides. AftB is essential for Mtb's survival and is deemed a potential new drug target for TB. Here we report the atomic-resolution structures of AftB obtained by cryo-EM in both apo and donor substrate analog bound forms. The apo structure revealed that AftB contains 11 transmembrane helices belonging to the GT-C glycosyltransferase superfamily and a periplasmic segment of a novel fold which might direct the binding of polysaccharides acceptor. AftB forms a large putative substrate-binding cavity, with one side facing the periplasmic domain and the other facing the lipid bilayer. The substrate-bound structure further unmasked the nature of the active site. Using synthetic AftB donor and acceptor substrates, we recapitulated the enzymatic reaction in vitro. And the functional analysis using AftB variants further proved the importance of the active site revealed from our structures. We proposed that AftB catalyzes the glycosylation reaction by utilizing a conserved aspartate residue as the base catalyst. Collectively, our work provides a structural basis for understanding the molecular mechanism of AftB and a blueprint for developing new drugs to combat the increasing threat caused by Mtb.