A Second, Druggable Binding Site in UDP-Galactopyranose Mutase from Mycobacterium tuberculosis?

UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.

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.

The nerve of ovulation-inducing factor in semen.

A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of ß nerve growth factor (ß-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as ß-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC(12)) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of ß-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is ß-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.

The structure of NtdA, a sugar aminotransferase involved in the kanosamine biosynthetic pathway in Bacillus subtilis, reveals a new subclass of aminotransferases.

NtdA from Bacillus subtilis is a sugar aminotransferase that catalyzes the pyridoxal phosphate-dependent equatorial transamination of 3-oxo-α-D-glucose 6-phosphate to form α-D-kanosamine 6-phosphate. The crystal structure of NtdA shows that NtdA shares the common aspartate aminotransferase fold (Type 1) with residues from both monomers forming the active site. The crystal structures of NtdA alone, co-crystallized with the product α-D-kanosamine 6-phosphate, and incubated with the amine donor glutamate reveal three key structures in the mechanistic pathway of NtdA. The structure of NtdA alone reveals the internal aldimine form of NtdA with the cofactor pyridoxal phosphate covalently attached to Lys-247. The addition of glutamate results in formation of pyridoxamine phosphate. Co-crystallization with kanosamine 6-phosphate results in the formation of the external aldimine. Only α-D-kanosamine 6-phosphate is observed in the active site of NtdA, not the ß-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VIß family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.

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

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.

A previously unrecognized kanosamine biosynthesis pathway in Bacillus subtilis.

The ntd operon in Bacillus subtilis is essential for biosynthesis of 3,3'-neotrehalosadiamine (NTD), an unusual nonreducing disaccharide reported to have antibiotic properties. It has been proposed that the three enzymes encoded within this operon, NtdA, NtdB, and NtdC, constitute a complete set of enzymes required for NTD synthesis, although their functions have never been demonstrated in vitro. We now report that these enzymes catalyze the biosynthesis of kanosamine from glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-dehydrogenase, NtdA is a pyridoxal phosphate-dependent 3-oxo-glucose-6-phosphate:glutamate aminotransferase, and NtdB is a kanosamine-6-phosphate phosphatase. None of these enzymatic reactions have been reported before. This pathway represents an alternate route to the previously reported pathway from Amycolatopsis mediterranei which derives kanosamine from UDP-glucose.

Towards the crystal structure elucidation of eukaryotic UDP-galactopyranose mutase.

UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. Eukaryotic UGMs from Aspergillus fumigatus and Leishmania major have been purified to homogeneity by means of Ni(2+)-affinity chromatography and crystallized. Eukaryotic UGM structure elucidation was not straightforward owing to high pseudo-symmetry, twinning and very low anomalous signal. Phasing to 2.8 Å resolution using SAD was successful for L. major UGM. However, the maps could only be improved by iterative density modification and manual model building. High pseudo-symmetry and twinning prevented correct space-group assignment and the completion of structure refinement. The structure of A. fumigatus UGM to 2.52 Å resolution was determined by molecular replacement using the incomplete 2.8 Å resolution L. major UGM model.

Structural investigation of myo-inositol dehydrogenase from Bacillus subtilis: implications for catalytic mechanism and inositol dehydrogenase subfamily classification.

Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxy group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild-type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary complexes with inositol and inosose. BsIDH is a tetramer, with a novel arrangement consisting of two long continuous ß-sheets, formed from all four monomers, in which the two central strands are crossed over to form the core of the tetramer. Each subunit in the tetramer consists of two domains: an N-terminal Rossmann fold domain containing the cofactor-binding site, and a C-terminal domain containing the inositol-binding site. Structural analysis allowed us to determine residues important in cofactor and substrate binding. Lys97, Asp172 and His176 are the catalytic triad involved in the catalytic mechanism of BsIDH, similar to what has been proposed for related enzymes and short-chain dehydrogenases. Furthermore, a conformational change in the nicotinamide ring was observed in some ternary complexes, suggesting hydride transfer to the si-face of NAD+. Finally, comparison of the structure and sequence of BsIDH with other putative inositol dehydrogenases allowed us to differentiate these enzymes into four subfamilies based on six consensus sequence motifs defining the cofactor- and substrate-binding sites.

Expression, purification and preliminary X-ray crystallographic analysis of UDP-galactopyranose mutase from Deinococcus radiodurans.

UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. A UGM-substrate complex from Deinococccus radiodurans has been expressed, purified and crystallized. Crystals were obtained by the microbatch-under-oil method at room temperature. The crystals diffracted to 2.36 A resolution at the Canadian Light Source. The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 134.0, b = 176.6, c = 221.6 A. The initial structure solution was determined by molecular replacement using UGM from Mycobacterium tuberculosis (PDB code 1v0j) as a template model.

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold.

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.

Aspergillus nidulans cell wall composition and function change in response to hosting several Aspergillus fumigatus UDP-galactopyranose mutase activity mutants.

Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a ß-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased ß-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced ß-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and ß-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.

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.

The structure of a putative S-formylglutathione hydrolase from Agrobacterium tumefaciens.

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 A resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the alpha/beta hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C--O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed.

Structure of Escherichia coli Lytic transglycosylase MltA with bound chitohexaose: implications for peptidoglycan binding and cleavage.

Crystal structures of an inactive mutant (D308A) of the lytic transglycosylase MltA from Escherichia coli have been determined in two different apo-forms, as well as in complex with the substrate analogue chitohexaose. The chitohexaose binds with all six saccharide residues in the active site groove, with an intact glycosidic bond at the bond cleavage center. Its binding induces a large reorientation of the two structural domains in MltA, narrowing the active site groove and allowing tight interactions of the oligosaccharide with residues from both domains. The structures identify residues in MltA with key roles in the binding and recognition of peptidoglycan and confirm that Asp-308 is the single catalytic residue, acting as a general acid/base. Moreover, the structures suggest that catalysis involves a high energy conformation of the scissile glycosidic linkage and that the putative oxocarbenium ion intermediate is stabilized by the dipole moment of a nearby alpha-helix.

Escherichia coli MltA: MAD phasing and refinement of a tetartohedrally twinned protein crystal structure.

Crystals were grown of a mutant form of the bacterial cell-wall maintenance protein MltA that diffracted to 2.15 A resolution. When phasing with molecular replacement using the native structure failed, selenium MAD was used to obtain initial phases. However, after MAD phasing the crystals were found to be tetartohedrally twinned, hampering correct space-group determination and refinement. A refinement protocol was designed to take tetartohedral twinning into account and was successfully applied to refine the structure. The refinement protocol is described and the reasons for the failure of molecular replacement and the success of MAD are discussed in terms of the effects of the tetartohedral twinning.

Purification, crystallization and preliminary X-ray analysis of the lytic transglycosylase MltA from Escherichia coli.

The lytic transglycosylase MltA from Escherichia coli with its membrane anchor and signal sequence deleted has been purified to homogeneity by means of cation-exchange chromatography. The enzyme was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 103.70, c = 109.84 A and one molecule per asymmetric unit. Crystals diffract to 2.2 A resolution on a synchrotron-radiation source.