Identification of the NAD(P)H Binding Site of Eukaryotic UDP-Galactopyranose Mutase

Abstract

UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP+. NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All twelve residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.

UDP-Galactopyranose Mutase in Nematodes

“…37,45,51-53 Recently, two independent groups applied protein x-ray crystallography to determine the structure and investigate a potential biological reducing agent of the A. fumigatus UGM. 54-56 These studies revealed that despite differences in the prokaryotic and A. fumigatus genes, the overall structure and folds of prokaryotic and eukaryotic UGMs are similar. 54,55 Our ability to trap a catalytic intermediate (compound 2 ) further suggested that the active site of CeUGM is similar to that of its prokaryotic homologs.…”

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

“…2 -NADPH was synthesized by the method of Jeong and Gready (27) with some minor modifications as previously described (28). Briefly, 25 mg of NADP ϩ (5.6 mM final concentration), 480 l of 2-propanol-d8 (1 M final concentration), and 50 units of alcohol dehydrogenase (T. brockii) were added to 6 ml of 25 mM Tris-Cl, pH 9.0.…”

Structure, mechanism, and dynamics of UDP-galactopyranose mutase

“…Eukaryotic UGMs from the fungus A. fumigatus and from the parasites T. cruzi , L. mexicana , and L. infantum have been shown to utilize NADPH as a redox partner (Table 1) [30, 37, 38]. In addition, these enzymes can stabilize the reduced form of the flavin, even under aerobic conditions.…”

“…The rate of oxidation is 200–1500-fold slower than the reduction step, thus, the enzymes can turn over several hundred times before becoming oxidized (inactivated) [30, 38]. The structures of A. fumigatus UGM in complex with NADP(H) have recently been solved and a novel NADPH binding domain was identified [37]. Thus, at least for eUGMs, the mechanism of activation by NAD(P)H has been established.…”