Conformational switching of Trp399 regulates opening and closing of loop-1 in galactofuranosyl transferase 2 of Mycobacterium tuberculosis.

Mycobacterial cell-wall assembly depends on multiple glycosyltransferases among which the processive galactofuranosyl transferase 2 (GlfT2) executes alternating β(1 → 5) and β(1 → 6) transfers of galactofuranose (Galf) within a single catalytic site through an oxocarbenium-like transition state. Despite the well characterized SN2-like chemistry of GlfT2, the role of loop-1 dynamics in regulating substrate binding and processivity remains poorly understood. Here, we modeled the ternary complex of GlfT2 with UDP-Galf and a galactan acceptor (n = 3) to show that loop-1 undergoes conformational gating governed by Trp399. This residue acts as a conformational pivot, stabilizing the acceptor through CH-π interactions in a compact closed state and facilitating the post-reaction rearrangement toward an open state compatible with acceptor translocation. These states are defined by distinct Trp399 side chain conformations and are separated by a shallow free energy difference (∼1 kcal mol-1), enabling dynamic interconversion. Markov state modeling shows that transitions between the closed and open loop-1 states occur on the nanosecond timescale, with a reactive mean first passage time of 42.5 ns. This rearrangement proceeds through both direct and an intermediate-mediated pathways, contributing 54.2% and 45.8% of the total reactive flux, respectively. Together, these findings support a mechanistic model in which Trp399-mediated loop-1 plasticity couples substrate stabilization with post-reaction rearrangement, providing new insight into the processive elongation mechanism of GlfT2.