Structural Insights into Mycobacteriales Galactan Biosynthesis

Abstract

The order Mycobacteriales includes a number of severe human pathogens, including Mycobacterium tuberculosis, the causative agent of tuberculosis and a leading cause of infectious disease-related mortality worldwide. The unique cell wall structure of these bacteria is essential for their viability, and has been studied as a potential target for novel therapeutics development. A key component of the mycobacterial cell wall is the galactan, a 30-40 residue linear polysaccharide of galactofuranose (Galf) with an alternating β(1,5) and β(1,6) linkage pattern, synthesized by the polymerase Galactofuranosyl Transferase 2 (GlfT2). While GlfT2 has been established as a processive polymerase with intrinsic sequence control, the mechanism underlying this activity remains unclear. In the studies presented here, we provide structural insights into Nocardia brasiliensis GlfT2 (NbrGlfT2) using X-ray crystallography and cryo-electron microscopy. We characterize both the acceptor-bound and membrane-embedded structures of NbrGlfT2 and propose three models for its catalysis: Processive Galactan Sliding, Feedback-Regulated Sequence Control, and Membrane Curvature-Mediated Polymerization. Furthermore, we structurally characterize a previously undescribed GlfT2 paralog from Rhodococcus equi, which we term ReqGlfT3. We confirm its galactofuranosyl transferase activity and identify the production of β(1,3) and β(1,5) linkages. These findings offer new insights into GlfT2 and related polymerizing glycosyltransferases, which will provide insights into enzymatic regioselectivity mechanisms and polysaccharide biosynthesis across the bacterial kingdom.