Structural Insight into the Unique Substrate Binding Mechanism and Flavin Redox State of UDP-galactopyranose Mutase from Aspergillus fumigatus

• Published in: The Journal of biological chemistry 2012-03, Vol.287 (14), p.10780-10790
• Main Authors: van Straaten, Karin E., Routier, Françoise H., Sanders, David A.R.
• Format: Article
• Language: English
• Subjects: Aspergillus fumigatus - enzymology; Carbohydrate-binding Protein; Catalytic Domain; Cell Wall; Conserved Sequence; Crystallography, X-Ray; Enzyme Mutation; Enzyme Stability; Enzyme Structure; Flavins - metabolism; Intramolecular Transferases - chemistry; Intramolecular Transferases - metabolism; Ligands; Membrane Biogenesis; Models, Molecular; Oxidation-Reduction; Protein Binding; Protein Structure and Folding; Uridine Diphosphate Galactose - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.322974

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). As in prokaryotic UGMs, the flavin needs to be reduced for the enzyme to be active. Here we present the first eukaryotic UGM structures from Aspergillus fumigatus (AfUGM). The structures are of UGM alone, with the substrate UDP-Galp and with the inhibitor UDP. Additionally, we report the structures of AfUGM bound to substrate with oxidized and reduced flavin. These structures provide insight into substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues Arg-182 and Arg-327. Comparison with prokaryotic UGM reveals that despite low sequence identity with known prokaryotic UGMs the overall fold is largely conserved. Structural differences between prokaryotic UGM and AfUGM result from inserts in AfUGM. A notable difference from prokaryotic UGMs is that AfUGM contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor. This loop flipping has not been observed in prokaryotic UGMs. In addition we have determined the crystals structures and steady-state kinetic constants of the reaction catalyzed by mutants R182K, R327K, R182A, and R327A. These results support our hypothesis that Arg-182 and Arg-327 play important roles in stabilizing the position of the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis. Background: UDP-galactopyranose mutase (UGM) is a critical enzyme for the proper formation of the cell wall of pathogenic microbes. Results: The structure of UGM in complex with substrate reveals novel features of substrate binding. Conclusion: Oxidation and reduction of the flavin cofactor causes rearrangements in the active site that affect substrate binding. Significance: These are the first structures of UGM from a eukaryotic pathogen.