Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Karr, Dale B.; Nix, Jay C.; Sobrado, Pablo; Tanner, John J.

doi:10.1074/jbc.m111.327536

Cited by 33 publications

(114 citation statements)

References 46 publications

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“…We note that this pattern of differential ligand occupancy (high in protomers A and B, low in protomers C and D) was observed in our studies of substrate and inhibitor binding to AfUGM, which also involved soaking the P 6 5 22 crystal form used here. 20 The maps allowed the building of complete models of NADPH at nearly full occupancy (q = 0.9) in chains A and B (Figure 2B). In chains C and D of the tetramer, the density was strong for the ADP group but weaker for the nicotinamide riboside group.…”

Section: Resultsmentioning

confidence: 99%

“…20–22 Briefly, the oxidation state of the FAD can be deduced from the conformation of the conserved histidine loop (Gly61-Gly62-His63), the identity of the hydrogen-bonding partner of the flavin N5 (Arg327 for FAD, Gly62 for FADH − ), the orientation of Trp315, and the curvature of the isoalloxazine (planar for FAD, bent by 7° for FADH − ).…”

Section: Resultsmentioning

confidence: 99%

“…Methods for the crystallization of oxidized AfUGM have been described; 20 see Supporting Information (SI) for details. Kinetic protein crystallography 26 was used to determine structures of oxidized AfUGM (AfUGM o ) complexed with NADPH or NADH, and reduced AfUGM (AfUGM r ) after dissociation of NADP + .…”

Section: Methodsmentioning

confidence: 99%

“…FAD and NADPH are colored yellow and pink, respectively. For reference, the AfUGM o -NADPH structure has been overlaid with the AfUGM r -UDP-Gal p complex (PDB code 3uth 20 ) and the UDP-Gal p (green) included in this image. The dashed yellow line denotes the 6.3 Å distance between the adenine and uracil sites.…”

Section: Figurementioning

confidence: 99%

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

Dhatwalia

Singh²,

Solano

et al. 2012

J. Am. Chem. Soc.

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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

Dhatwalia

Singh²,

Solano

et al. 2012

J. Am. Chem. Soc.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Conformational Control of UDP-Galactopyranose Mutase Inhibition

et al. 2017

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UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5′-diphosphate-α-D-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies to optimize inhibitor potency by exploiting UGM conformational flexibility.

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Implicit vs. Explicit Solvent Models for Calculating X‐ray Solution Scattering Curves^#

Ganesan

Kim

Lee

et al. 2015

Bulletin Korean Chem Soc

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Theoretical calculation of X‐ray solution scattering curves of proteins in the solution phase is strongly influenced by solvent contributions in the form of solvent‐excluded volume and hydration layer that are generally represented either implicitly or explicitly. To investigate the effect of the implicit and explicit solvent models on the calculated scattering curves, we developed a new program, X‐ray Solution Scattering (XSoS) based on implicit (XSoS‐implicit) and explicit (XSoS‐explicit) solvent models. Both XSoS‐implicit and XSoS‐explicit can calculate X‐ray solution scattering curves with high accuracy. Overall, the implicit solvent model has practical advantages over the explicit solvent model for the analysis of experimental X‐ray solution scattering data.

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Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

Cited by 33 publications

References 46 publications

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

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

Conformational Control of UDP-Galactopyranose Mutase Inhibition

Implicit vs. Explicit Solvent Models for Calculating X‐ray Solution Scattering Curves^#

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Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

Cited by 33 publications

References 46 publications

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

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

Conformational Control of UDP-Galactopyranose Mutase Inhibition

Implicit vs. Explicit Solvent Models for Calculating X‐ray Solution Scattering Curves#

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Implicit vs. Explicit Solvent Models for Calculating X‐ray Solution Scattering Curves^#