Structural insight into the unique substrate binding mechanism and flavin redox state of UDP-galactopyranose mutase from Aspergillus fumigatus

Abstract

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.

FIGURE 1.

A, stereodiagram of the monomer from AfUGM, with numbering of the helices and sheets. The numbers correspond to the labels in supplemental Fig. S1, a structure-based sequence alignment. Domain 1 is colored blue, domain 2 is colored green, and domain 3 is colored black. The mobile loops are colored yellow and the additional inserts in AfUGM are colored red. The FAD and UDP-Galp are shown as ball-and-stick representations. B, ribbon representation of reduced AfUGM·UDP-Galp tetramer. Individual subunits are colored red, green, yellow, and blue. FADH₂ and UDP-Galp are shown in stick representation. C, superposition of unliganded AfUGM (blue) and reduced AfUGM·UDP-Galp complex (green). Open conformation of mobile loops I and II are shown in yellow. Closed conformation of mobile loops I and II shown in red. The two conformations of loop III are colored the same as for the mobile loops. Arg-182, FADH₂, and UDP-Galp are shown in stick representation. D, overall monomer structure of reduced AfUGM·UDP-Galp shown as a ribbon representation (left) and overall monomer structure of reduced DrUGM·UDP-Galp (right). Coloring scheme is the same as for A.

FIGURE 2.

A, close-up of active sites from unliganded (left) and liganded AfUGM (right). Unliganded AfUGM, mobile loop III (residues 59–66) above the isoalloxazine ring with His-63 pointing into the highly conserved hydrophobic pocket. Arg-327 is at hydrogen bonding distance from N5 and O4 of FAD. Mobile loop III in the reduced AfUGM·UDP-Galp complex has flipped. His-63 sits above N5 of FADH₂. Arg-327 is at hydrogen bonding distance of UDP-Galp. The F_o − F_c-electron density of the ligand (contoured at 3σ) is shown as a green wireframe in the right panel. B, stereodiagram comparison of the active sites from AfUGM·UDP-Galp (green) and DrUGM·UDP-Galp (blue). Labeling of active site residues is according to AfUGM sequence. C, stereodiagram of AfUGM active site with bound UDP-Galp. Residues shown in thick lines are within 4 Å from UDP-Galp. Residues in red are from closed mobile loops I and II. Residues in green (90–114) help to stabilize loop II and in positioning the uracil portion of UDP-Galp. D, overlay of reduced AfUGM·UDP-Galp (blue) on AfUGM·UDP (red). The binding mode of UDP is the same as the binding mode of the UDP moiety in the reduced UDP-Galp crystal structure. The only notable difference with the reduced UDP-Galp complex structure is that upon oxidation of the flavin the Phe-66 side chain moved outwards the active site and loop III at N3 side of the isoalloxazine ring moved ∼1 Å down.

FIGURE 3.

Binding mode of UDP-Galp in arginine mutants. A, superposition of R327A AfUGM with bound UDP-Galp (blue) on reduced AfUGM·UDP-Galp (red). UDP-Galp is bound in a non-productive binding mode in R327A AfUGM. Arg-182 is in the same position as in wild type AfUGM. B, the R182K AfUGM mutant with bound UDP-Galp. Only the UDP moiety is visible and no density for Galp moiety. R182K is within 3.6 Å from the Asn-457 side chain but has no interaction with the phosphates or the galactose moiety. Phe-66 points out of the active site. C, R327K AfUGM with bound UDP-Galp. NZ of R327K is in the same position as NH1 of Arg-327 and makes the same interactions with galactose moiety and the β-phosphate as in wild type complex. Phe-66 points into active site.

FIGURE 4.

Overlay of reduced AFUGM·UDP-Galp (blue) and non-reduced AfUGM·UDP-Galp (red). The interactions in the sugar binding site are significantly different in the non-reduced UDP-Galp complex structure.