David A. R. Sanders

UDP-galactopyranose mutase has a novel structure and mechanism.

Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi. UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase). The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target. The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear. We have solved the crystal structure of Escherichia coli mutase to 2.4 Å resolution. The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues. Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD. Assay results establish that the enzyme is active only when flavin is reduced. We conclude that mutase most likely functions by transient reduction of substrate.

Aspergillus nidulans UDP-galactopyranose mutase, encoded by ugmA plays key roles in colony growth, hyphal morphogensis, and conidiation

Growing resistance to current anti-fungal drugs is spurring investigation of new targets, including those in fungal wall metabolism. Galactofuranose (Galf) is found in the cell walls of many fungi including Aspergillus fumigatus, which is currently the most prevalent opportunistic fungal pathogen in developed countries, and A. nidulans, a closely-related, tractable model system. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose into UDP-Galf prior to incorporation into the fungal wall. We deleted the single-copy UGM sequence (AN3112.4, which we call ugmA) from an A. nidulans nkuADelta strain, creating ugmADelta. Haploid ugmADelta strains were able to complete their asexual life cycle, showing that ugmA is not essential. However, ugmADelta strains had compact colonial growth, which was associated with substantially delayed and abnormal conidiation. Compared to a wildtype morphology strain, ugmADelta strains had aberrant hyphal morphology, producing wide, uneven, highly-branched hyphae, with thick, relatively electron-dense walls as visualized by transmission electron microscopy. These effects were partially remediated by growth on high osmolarity medium, or on medium containing 10 microg/mL Calcofluor, consistent with Galf being important in cell wall structure and/or function.

Structural basis of substrate binding to UDP-galactopyranose mutase: crystal structures in the reduced and oxidized state complexed with UDP-galactopyranose and UDP.

D-Galactofuranose (Galf) residues are found in the cell walls of pathogenic microbes such as Mycobacterium tuberculosis, and are essential for viability. UDP-galactopyranose mutase (UGM) is a unique flavo-enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). UDP-Galf is the active precursor of Galf residues found in cell walls. Despite the wealth of biochemical/mechanistic data generated for UGM, the structural basis of substrate binding is still lacking. Here, we report the crystal structures of UGM from Deinococcus radiodurans (drUGM) in complex with its natural substrate (UDP-Galp) and UDP. Crystal structures of drUGM:UDP-Galp complexes with oxidized and reduced FAD were determined at 2.36 A and 2.50 A resolution, respectively. The substrate is buried in the active site in an unusual folded conformation and the anomeric carbon of the galactose is at a favorable distance (2.8 A) from N5 of FAD to form an FAD-galactose adduct. The mobile loops in the substrate complex structure exist in a closed conformation. The drUGM-UDP complex structure was determined at 2.55 A resolution and its overall structure is identical with that of the oxidized and reduced complexes, including the conformation of the mobile loops. Comparison with the recently reported UGM:UDP-glucose complex structure reveals key differences and the structures reported here are likely to represent the productive/active conformation of UGM. These structures provide valuable insights into substrate recognition and a basis for understanding the mechanism. These complex structures may serve as a platform for structure-guided design of inhibitors of UGM.

Dissection of complex protein dynamics in human thioredoxin

We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47–0.67 and 10.8–13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10–17.5 ps for three mutants. A robust dynamical process in 95–114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275–615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeA Delta strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmA Delta strain. As for the ugmA Delta strain, ugeA Delta colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeA Delta strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeA Delta, and ugmA Delta hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmA Delta strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-D-galactopyranose into UDP-α-D-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-D-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.

Structural basis of ligand binding to UDP-galactopyranose mutase from Mycobacterium tuberculosis using substrate and tetrafluorinated substrate analogues.

UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His6-MBP-tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxy-tetrafluorinated analogues of both UDP-Galp (UDP-F4-Galp) and UDP-Galf (UDP-F4-Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogues bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show an overall binding mode similar to those reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active-site closure. In addition, the complex structure of MtUGM with UDP-F4-Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high-energy conformation ((4)E) within the catalytic pocket. Moreover, these investigations provide structural insights into the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogues. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities for the use of polyfluorination in the design of carbohydrate mimetics.

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

Galactofuranose (Galf) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Galf from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Galf residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Galf transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. AfglfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Galf, which was restored in the AfglfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.

Antimycobacterial activity of UDP-galactopyranose mutase inhibitors

The galactofuran region of the mycobacterial cell wall consists of alternating 5- and 6-linked beta-d-galactofuranose (beta-D-Galf) residues, essential for viability. UDP-galactofuranose (UDP-Galf), the donor for Galf, is synthesised from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (UGM), which is not found in humans, rendering it a therapeutic target. The in vitro properties, i.e. enzymatic activity, antimycobacterial activity, cellular toxicity, activity in mycobacterial-infected macrophages and activity against non-replicating persistent mycobacteria, of (4-chlorophenyl)-[1-(4-chlorophenyl)-3-hydroxy-5-methyl-1H-pyrazol-4-yl]-methanone and 3-(4-iodophenyl)-2-[4-(3,4-dichlorophenyl)-thiazol-2-ylamino]-propionic acid were studied. The former compound, a pyrazole, was an inhibitor of UGM from Mycobacterium tuberculosis and Klebsiella pneumoniae and was effective against Mycobacterium smegmatis, Mycobacterium bovis BCG and M. tuberculosis but ineffective against other bacterial strains tested. This compound showed potency against mycobacteria in infected macrophages but exhibited moderate cellular toxicity and was ineffective against non-replicating persistent mycobacteria. This is the first report of a compound both with UGM inhibitory properties and broad antimycobacterial activities. The latter compound, an aminothiazole, was active against UGM from K. pneumoniae and M. tuberculosis but was ineffective against M. bovis BCG or M. tuberculosis as well as demonstrating higher cellular toxicity. These data validate the choice of UGM as a target for active antimycobacterial therapy and confirm the pyrazole compound as a viable lead candidate.

Succinylphosphonate Esters Are Competitive Inhibitors of MenD That Show Active-Site Discrimination between Homologous α-Ketoglutarate-Decarboxylating Enzymes

MenD is a thiamin diphosphate-dependent enzyme catalyzing the first unique step in menaquinone biosynthesis in bacteria. We have synthesized acylphosphonate ester analogues of alpha-ketoglutarate, a substrate of MenD. These compounds are competitive inhibitors of MenD, with K(i) values as low as 700 nM. Observed structure-activity relationships are in notable contrast to those reported previously for succinylphosphonate inhibition of the alpha-ketoglutarate dehydrogenase complex, despite the apparent homology of these enzymes, and the identical decarboxylation reactions catalyzed. Inhibiting menaquinone biosynthesis is a proposed approach to inhibiting Mycobacterium tuberculosis growth. These inhibitors showed no significant inhibition of M. tuberculosis growth in vitro under aerobic and hypoxic conditions but give new information about the binding characteristics of the MenD active site. Site-directed mutation of Ser391 to alanine had only a minor effect on catalysis, but even the conservative mutation of Arg395 to lysine had a significant effect on the catalytic processing of isochorismate.