How Mycobacterium tuberculosis Galactofuranosyl Transferase 2 (GlfT2) Generates Alternating β-(1-6) and β-(1-5) Linkages: A QM/MM Molecular Dynamics Study of the Chemical Steps.

Mycobacterium tuberculosis features a unique cell wall that protects the bacterium from the external environment. Disruption of the cell wall assembly is a promising direction for novel anti-tuberculotic drugs. A key component of the cell wall is galactan, a polysaccharide chain composed of galactofuranose (Galf) units connected by alternating β-(1-5) and β-(1-6) linkages. The majority of the galactan chain is biosynthesized by a bifunctional enzyme-galactofuranosyl transferase 2 (GlfT2). GlfT2 catalyzes two reactions: the formation of β-(1-5) and β-(1-6) linkages. It was suggested that the enzyme acts through a processive mechanism until it adds 30-35 Galf units in a single active site. We applied a QM/MM string method coupled with ab initio molecular dynamics simulations to study the two reactions catalyzed by GlfT2. We showed that both reactions proceed very similarly and feature similar transition-state structures. We also present novel information about the ring puckering behavior of the five-membered furanose ring during the glycosyltransferase reaction and a calculated transition-state structure with galactose in a furanose form that may be used as a guide for the rational design of very specific and extremely potent inhibitors, that is, transition-state analogues, for GlfT2. Due to the absence of a furanose form of galactose in humans, transition-state-analogous inhibitors represent an attractive scaffold for the development of novel antibacterial drugs.