Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

Abstract

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

Structural Basis of Ligand Binding to UDP-Galactopyranose Mutase from Mycobacterium tuberculosis Using Substrate and Tetrafluorinated Substrate Analogues

“…53, 54 However, in spite of many efforts, none of these UGMs could be crystallized with UDP-Galf or furanoside analogs. Therefore, the binding mode of UGM and its substrate UDP-Galf has remained elusive.…”

Conformational Control of UDP-Galactopyranose Mutase Inhibition

“…Our structure of oxidized GSG-CdUGM in complex with UDP indicates the inactive enzyme can adopt a closed conformation analogous to that observed for the catalytically competent KpUGM. Other closed UGM orthologs have been crystallized with oxidized FAD either with or without bound substrates, including DrUGM 30 , EcUGM, 3 AfUGM 51 , and TcUGM 34 . These findings suggest that the energy barrier for conformational change is small, regardless of the FAD oxidation state.…”

“…32 Eukaryotic UGMs also adopt multiple conformations, and they have a second mobile flap containing an asparagine residue involved in substrate recognition. 33, 34 Additionally, molecular dynamics studies indicate a third mobile loop near the active site in the Trypanosoma cruzi UGM (TcUGM). 34 Dramatic conformational changes are also observed in a histidine-containing loop in eukaryotic UGMs.…”

“…33, 34 Additionally, molecular dynamics studies indicate a third mobile loop near the active site in the Trypanosoma cruzi UGM (TcUGM). 34 Dramatic conformational changes are also observed in a histidine-containing loop in eukaryotic UGMs. 33, 34 These observations highlight the flexibility of UGMs and suggest that progress in understanding UGM conformational variation would advance inhibitor design.…”

“…34 Dramatic conformational changes are also observed in a histidine-containing loop in eukaryotic UGMs. 33, 34 These observations highlight the flexibility of UGMs and suggest that progress in understanding UGM conformational variation would advance inhibitor design.…”