Biophysical and biochemical characterization of the thioredoxin system from Colwellia psychrerythraea.

The thioredoxin system is a ubiquitous oxidoreductase system consisting of the enzyme thioredoxin reductase, the protein thioredoxin, and the cofactor nicotinamide adenine dinucleotide phosphate. The system has been comprehensively studied from many organisms, such as Escherichia coli; however, structural and functional analysis of this system from psychrophilic bacteria has not been as extensive. In this study, the thioredoxin system proteins of a psychrophilic bacterium, Colwellia psychrerythraea, were characterized using biophysical and biochemical techniques. Analysis of the complete genome sequence of the C. psychrerythraea thioredoxin system suggested the presence of a putative thioredoxin reductase and at least three thioredoxin. In this study, these identified putative thioredoxin system components were cloned, overexpressed, purified, and characterized. Our studies have indicated that the thioredoxin system proteins from E. coli were more stable than those from C. psychrerythraea. Consistent with these results, kinetic assays indicated that the thioredoxin reductase from E. coli had a higher optimal temperature than that from C. psychrerythraea.

Unraveling the unexpected aggregation behavior of Pyrazole-Based compounds Targeting Mycobacterium tuberculosis UDP-Galactopyranose mutase.

A pyrazole-based compound, MS208, was previously identified as an inhibitor of UDP-Galactopyranose Mutase from Mycobacterium tuberculosis (MtUGM). Targeting this enzyme is a novel therapeutic strategy for the development of new antituberculosis agents because MtUGM is an essential enzyme for the bacterial cell wall synthesis and it is not present in human. It was proposed that MS208 targets an allosteric site in MtUGM as MS208 followed a mixed inhibition model. DA10, an MS208 analogue, showed competitive inhibition rather than mixed inhibition. In this paper, we have used an integrated biophysical approach, including thermal shift assays, dynamic light scattering and nuclear magnetic resonance experiments, to show that MS208 and many analogues displayed unexpected aggregation behavior against MtUGM.

Pyrazole and Triazole Derivatives as Mycobacterium tuberculosis UDP-Galactopyranose Inhibitors.

UDP-galactopyranose mutase (UGM) is an essential enzyme involved in the bacterial cell wall synthesis, and is not present in mammalian cells. Thus, UGM from Mycobacterium tuberculosis (Mtb) represents a novel and attractive drug target for developing antituberculosis agents. A pyrazole-based compound, MS208, was previously identified as a mixed inhibitor of MtbUGM which targets an allosteric site. To understand more about the structure activity relationship around the MS208 scaffold as a MtbUGM inhibitor, thirteen pyrazoles and triazole analogues were synthesized and tested against both MtbUGM and Mycobacterium tuberculosis in vitro. While the introduced structural modifications to MS208 did not improve the antituberculosis activity, most of the compounds showed MtbUGM inhibitory activity. Interestingly, the pyrazole derivative DA10 showed a competitive model for MtbUGM inhibition with improved Ki value of 51 ± 4 µM. However, the same compound did not inhibit the growth of Mycobacterium tuberculosis.

Snapshots along the catalytic path of KabA, a PLP-dependent aminotransferase required for kanosamine biosynthesis in Bacillus cereus UW85.

Kanosamine is an antibiotic and antifungal monosaccharide. The kanosamine biosynthetic pathway from glucose 6-phosphate in Bacillus cereus UW85 was recently reported, and the functions of each of the three enzymes in the pathway, KabA, KabB and KabC, were demonstrated. KabA, a member of a subclass of the VIß family of PLP-dependent aminotransferases, catalyzes the second step in the pathway, generating kanosamine 6-phosphate (K6P) using l-glutamate as the amino-donor. KabA catalysis was shown to be extremely efficient, with a second-order rate constant with respect to K6P transamination of over 107 M-1s-1. Here we report the high-resolution structure of KabA in both the PLP- and PMP-bound forms. In addition, co-crystallization with K6P allowed the structure of KabA in complex with the covalent PLP-K6P adduct to be solved. Co-crystallization or soaking with glutamate or 2-oxoglutarate did not result in crystals with either substrate/product. Reduction of the PLP-KabA complex with sodium cyanoborohydride gave an inactivated enzyme, and crystals of the reduced KabA were soaked with the l-glutamate analog glutarate to mimic the KabA-PLP-l-glutamate complex. Together these four structures give a complete picture of how the active site of KabA recognizes substrates for each half-reaction. The KabA structure is discussed in the context of homologous aminotransferases.

Deep Learning Architecture to Assist With Steering a Powered Wheelchair.

This paper describes a novel Deep Learning architecture to assist with steering a powered wheelchair. A rule-based approach is utilized to train and test a Long Short Term Memory (LSTM) Neural Network. It is the first time a LSTM has been used for steering a powered wheelchair. A disabled driver uses a joystick to provide desired speed and direction, and the Neural Network provides a safe direction for the wheelchair. Results from the Neural Network are mixed with desired speed and direction to avoid obstacles. Inputs originate from a joystick and from three ultrasonic transducers attached to the chair. The resultant course is a blend of desired directions and directions that steer the chair to avoid collision. A rule-based approach is used to create a training and test set for the Neural Network system and applies deep learning to predict a safe route for a wheelchair. The user can over-ride the new system if necessary.

Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.

Structure and function of the putative thioredoxin 1 from the thermophilic eubacterium Thermosipho africanus strain TCF52B.

The thioredoxin system is a ubiquitous oxidoreductase system that consists of the enzyme thioredoxin reductase (TrxR), its cofactor nicotinamide adenine dinucleotide phosphate (NAD(P)H) and the protein thioredoxin (Trx). The system has been comprehensively studied from many organisms, such as Escherichia coli (E. coli); however, structural and functional analysis of this system from thermophilic bacteria has not been as extensive. In this study, Thermosipho africanus, a thermophilic eubacterium, Trx1 (TaTrx1) was successfully cloned, overexpressed and purified, to greater than 95% purity. Inspection of the amino acid sequence of TaTrx1 categorized the protein as a putative Trx. Its ability to reduce the interchain disulfides of insulin, in the presence of dithiothreitol, provided further evidence to suggest that it was a Trx. The three dimensional structure of the protein, determined using X-ray crystallography, provided additional evidence for this. The crystal structure was solved in space group P212121 to 1.8 A resolution and showed the characteristic thioredoxin fold; four ß-strands surrounded by three α-helices. The active site of TaTrx1 contained two cysteines that formed a disulfide bridge, and was structurally similar to the active site of EcTrx1. Further studies indicated that TaTrx1 was far more stable than Trx1 of E. coli (EcTrx1). The protein could withstand both higher temperatures and higher concentrations of guanidine hydrochloride before denaturing. Our studies have therefore identified a novel thermophilic putative Trx that structurally and functionally behaves like a Trx.

Selecting a Best Compromise Direction for a Powered Wheelchair Using PROMETHEE.

Research is described that determines the direction that a powered wheelchair will take, using the preference ranking organization method for enrichment of evaluations II (PROMETHEE II). This is the first time that this sort of decision making has been used in this type of application. A user suggests a desired direction and speed, and the decision-making system suggests a safe direction for a wheelchair. The two are mixed, and the wheelchair then tends to avoid obstacles. The inputs come from a powered wheelchair joystick and two ultrasonic transducers fixed onto the wheelchair, and the final direction is a simulation of the desired direction and a direction that moves the wheelchair away from obstacles. The arrangement assists a disabled driver to steer their wheelchairs. It uses a systematic decision-making process, and this paper presents the process and a new way of selecting a best compromise direction. Sensitivity analysis is employed to explore potentially suitable directions as uncertainty and risk may be present. A suitable direction is selected that provides a robust solution. The user would be able to override the suggestions by the PROMETHEE II system by holding the joystick in a position.

myo-Inositol dehydrogenase and scyllo-inositol dehydrogenase from Lactobacillus casei BL23 bind their substrates in very different orientations.

Many bacteria can use myo-inositol as the sole carbon source using enzymes encoded in the iol operon. The first step is catalyzed by the well-characterized myo-inositol dehydrogenase (mIDH), which oxidizes the axial hydroxyl group of the substrate to form scyllo-inosose. Some bacteria, including Lactobacillus casei, contain more than one apparent mIDH-encoding gene in the iol operon, but such redundant enzymes have not been investigated. scyllo-Inositol, a stereoisomer of myo-inositol, is not a substrate for mIDH, but scyllo-inositol dehydrogenase (sIDH) enzymes have been reported, though never observed to be encoded within the iol operon. Sequences indicate these enzymes are related, but the structural basis by which they distinguish their substrates has not been determined. Here we report the substrate selectivity, kinetics, and high-resolution crystal structures of the proteins encoded by iolG1 and iolG2 from L. casei BL23, which we show encode a mIDH and sIDH, respectively. Comparison of the ternary complex of each enzyme with its preferred substrate reveals the key variations allowing for oxidation of an equatorial versus an axial hydroxyl group. Despite the overall similarity of the active site residues, scyllo-inositol is bound in an inverted, tilted orientation by sIDH relative to the orientation of myo-inositol by mIDH.

Analysis of plant UDP-arabinopyranose mutase (UAM): Role of divalent metals and structure prediction.

UDP-arabinopyranose mutase (UAM) is a plant enzyme which interconverts UDP-arabinopyranose (UDP-Arap; a six-membered sugar) to UDP-arabinofuranose (UDP-Araf; a five-membered sugar). Plant mutases belong to a small gene family called Reversibly Glycosylated Proteins (RGPs). So far, UAM has been identified in Oryza sativa (Rice), Arabidopsis thaliana and Hordeum vulgare (Barley). The enzyme requires divalent metal ions for catalytic activity. Here, the divalent metal ion dependency of UAMs from O. sativa (rice) and A. thaliana have been studied using HPLC-based kinetic assays. It was determined that UAM from these species had the highest relative activity in a range of 40-80µM Mn2+. Excess Mn2+ ion concentration decreased the enzyme activity. This trend was observed when other divalent metal ions were used to test activity. To gain a perspective of the role played by the metal ion in activity, an ab initio structural model was generated based on the UAM amino acid sequence and a potential metal binding region was identified. Based on our results, we propose that the probable role of the metal in UAM is stabilizing the diphosphate of the substrate, UDP-Arap.

Using Self-Reliance Factors to Decide How to Share Control Between Human Powered Wheelchair Drivers and Ultrasonic Sensors.

A shared-control scheme for a powered wheelchair is presented. The wheelchair can be operated by a wheelchair driver using a joystick, or directed by a sensor system, or control can be combined between them. The wheelchair system can modify direction depending on the local environment. Sharing the control allows a disabled wheelchair driver to drive safely and efficiently. The controller automatically establishes the control gains for the sensor system and the human driver by calculating a self-reliance factor for the wheelchair driver. The sensor system can influence the motion of the wheelchair to compensate for some deficiency in a disabled driver. Practical tests validate the proposed techniques and designs.

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.

Putative thioredoxin Trx1 from Thermosipho africanus strain TCF52B: expression, purification and structural determination using S-SAD.

Thioredoxin is a small ubiquitous protein that plays a role in many biological processes. A putative thioredoxin, Trx1, from Thermosipho africanus strain TCF52B, which has low sequence identity to its closest homologues, was successfully cloned, overexpressed and purified. The protein was crystallized using the microbatch-under-oil technique at 289 K in a variety of conditions; crystals grown in 0.2 M MgCl2, 0.1 M bis-tris pH 6.5, 25%(w/v) PEG 3350, which grew as irregular trapezoids to maximum dimensions of 1.2 × 1.5 × 0.80 mm, were used for sulfur single-wavelength anomalous dispersion analysis. The anomalous sulfur signal could be detected to 2.83 Šresolution using synchrotron radiation on the 08B1-1 beamline at the Canadian Light Source. The crystals belonged to space group P212121, with unit-cell parameters a = 40.6, b = 41.5, c = 56.4 Å, α = ß = γ = 90.0°.

Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni.

Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and ß-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development.

Mechanism-based candidate inhibitors of uridine diphosphate galactopyranose mutase (UGM).

Uridine diphosphate-galactopyranose mutase (UGM), an enzyme found in many eukaryotic and prokaryotic human pathogens, catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), the latter being used as the biosynthetic precursor of the galactofuranose polymer portion of the mycobacterium cell wall. We report here the synthesis of a sulfonium and selenonium ion with an appended polyhydroxylated side chain. These compounds were designed as transition state mimics of the UGM-catalyzed reaction, where the head groups carrying a permanent positive charge were designed to mimic both the shape and positive charge of the proposed galactopyranosyl cation-like transition state. An HPLC-based UGM inhibition assay indicated that the compounds inhibited about 25% of UGM activity at 500 µM concentration.

Synthesis and biological evaluation of nonionic substrate mimics of UDP-Galp as candidate inhibitors of UDP galactopyranose mutase (UGM).

The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(ß-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds.

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.

Tyrosine 110 plays a critical role in regulating the allosteric inhibition of Campylobacter jejuni dihydrodipicolinate synthase by lysine.

Dihydrodipicolinate synthase (DHDPS), an enzyme found in most bacteria and plants, controls a critical step in the biosynthesis of l-lysine and meso-diaminopimelate, necessary components for bacterial cell wall biosynthesis. DHDPS catalyzes the condensation of pyruvate and (S)-aspartate-ß-semialdehyde, forming an unstable product that is dehydrated to dihydrodipicolinate. The tetrameric enzyme is allosterically inhibited by l-lysine, and a better understanding of the allosteric inhibition mechanism is necessary for the design of potent antibacterial therapeutics. Here we describe the high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites. These structures reveal a role for Y110 in the regulation of the allosteric inhibition by lysine. Mutation of Y110 to phenylalanine results in insensitivity to lysine inhibition, although the mutant crystal structure reveals that lysine does bind in the allosteric site. Comparison of the lysine-bound Y110F structure with wild-type structures reveals that key structural changes due to lysine binding are absent in this mutant.

Purification, crystallization and room-temperature X-ray diffraction of inositol dehydrogenase LcIDH2 from Lactobacillus casei BL23.

Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Šresolution.

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.