UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyses the reversible conversion of UDP-Galactopyranose (UDP-Galp) to UDP-Galactofuranose (UDPGalf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His 6 -MBP tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxytetrafluorinated analogs of both UDP-Galp (UDP-F 4 -Galp) and UDP-Galf (UDP-F 4 -Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogs bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show a similar overall binding mode as reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active site closure. In addition, the complex structure of MtUGM with UDP-F 4 -Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high energy conformation ( 4 E) within the catalytic pocket. Moreover, these investigations provide structural insights to the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogs. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities on the use of polyfluorination in the design of carbohydrate mimetics.

This study reports a novel class of inhibitors of uridine 5'-diphosphate (UDP) galactopyranose mutase (UGM) derived from a screening of natural products. This enzyme is an essential biocatalyst involved in the cell wall biosynthesis of Mycobacterium tuberculosis. Flavonoids are potent inhibitors of UGM. The synthesis of novel methylated flavonoids allowed a structure-activity relationship analysis to be performed and which functional groups and structural elements were required for UGM inhibition could be determined. The binding mode of one of the best inhibitors was found to be noncompetitive. Docking simulations indicated that this molecule was likely to bind UGM in its open conformation, in a cavity recently identified as a "druggable" pocket. Importantly, two of the best inhibitors of the M. tuberculosis UGM displayed moderate activity against whole M. tuberculosis cells. This study reports the first natural products that act as inhibitor of UGM. Given the importance of natural products in medicinal chemistry, these results create new opportunities for the discovery of new antitubercular agents.

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

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