RESUMEN
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.
Asunto(s)
Alteromonadaceae , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Proteínas Bacterianas/química , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/química , Transaminasas/química , Transaminasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Coenzimas/metabolismo , Cristalografía por Rayos X , Glucosamina/biosíntesis , Glutaratos/química , Glutaratos/metabolismo , Lisina/metabolismo , Modelos Moleculares , Conformación Proteica , Fosfato de Piridoxal/metabolismo , Transaminasas/genética , Transaminasas/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Asparagina/genética , Campylobacter jejuni/enzimología , Hidroliasas/genética , Estructura Cuaternaria de Proteína , Regulación Alostérica/genética , Asparagina/química , Hidroliasas/química , Hidroliasas/ultraestructuraRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Eubacterium , Tiorredoxinas/química , Secuencia de Aminoácidos , Insulina/química , Conformación Proteica , Estabilidad ProteicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos , Lacticaseibacillus casei/enzimología , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Proteínas Bacterianas/genética , Catálisis , Clonación Molecular , Cristalización , Cristalografía por Rayos X , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Inositol/metabolismo , Lacticaseibacillus casei/genética , Operón , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genéticaRESUMEN
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.
Asunto(s)
Arabidopsis/enzimología , Transferasas Intramoleculares/química , Oryza/enzimología , Azúcares de Uridina Difosfato/química , Sitios de Unión , Catálisis , Pared Celular/enzimología , Regulación de la Expresión Génica de las Plantas , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Iones/química , Cinética , Metales/química , Unión Proteica , Azúcares de Uridina Difosfato/metabolismoRESUMEN
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.â 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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Transferasas Intramoleculares/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Pirazoles/farmacología , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Transferasas Intramoleculares/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Pirazoles/química , Relación Estructura-ActividadRESUMEN
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 289â K in a variety of conditions; crystals grown in 0.2â M MgCl2, 0.1â M bis-tris pH 6.5, 25%(w/v) PEG 3350, which grew as irregular trapezoids to maximum dimensions of 1.2 × 1.5 × 0.80â mm, were used for sulfur single-wavelength anomalous dispersion analysis. The anomalous sulfur signal could be detected to 2.83â Å resolution 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 = 56.4â Å, α = ß = γ = 90.0°.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Tiorredoxinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Conformación Proteica , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
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.
Asunto(s)
Biomimética , Campylobacter jejuni/enzimología , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Regulación Alostérica , Cristalografía por Rayos X , Inhibidores Enzimáticos/químicaRESUMEN
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.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Galactosa/análogos & derivados , Isomerasas/antagonistas & inhibidores , Uridina Difosfato/análogos & derivados , Biocatálisis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Galactosa/metabolismo , Hidroxilación , Isomerasas/metabolismo , Mycobacterium tuberculosis/enzimología , Compuestos de Selenio/síntesis química , Compuestos de Selenio/química , Compuestos de Selenio/farmacología , Compuestos de Sulfonio/síntesis química , Compuestos de Sulfonio/química , Compuestos de Sulfonio/farmacología , Uridina Difosfato/metabolismoRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Galactosa/metabolismo , Transferasas Intramoleculares/antagonistas & inhibidores , Monosacáridos/farmacología , Uridina Difosfato/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Transferasas Intramoleculares/metabolismo , Conformación Molecular , Monosacáridos/síntesis química , Monosacáridos/química , Mycobacterium tuberculosis/enzimología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hidrocarburos Fluorados/farmacología , Transferasas Intramoleculares/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Uridina Difosfato Glucosa/farmacología , Sitios de Unión/efectos de los fármacos , Inhibidores Enzimáticos/química , Hidrocarburos Fluorados/química , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Ligandos , Estructura Molecular , Especificidad por Sustrato/efectos de los fármacos , Uridina Difosfato Glucosa/químicaRESUMEN
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.
Asunto(s)
Campylobacter jejuni/enzimología , Inhibidores Enzimáticos/farmacología , Hidroliasas/química , Hidroliasas/metabolismo , Lisina/farmacología , Tirosina/metabolismo , Regulación Alostérica/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Hidroliasas/antagonistas & inhibidores , Hidroliasas/genética , Ligandos , Lisina/metabolismo , Modelos Moleculares , Movimiento , Mutagénesis Sitio-Dirigida , MutaciónRESUMEN
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 77â 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 1.6â Å resolution.
Asunto(s)
Proteínas Bacterianas/química , Lacticaseibacillus casei/química , Deshidrogenasas del Alcohol de Azúcar/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Lacticaseibacillus casei/enzimología , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismoRESUMEN
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 AnugmAâµ 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 AnugmAâµ. 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 AnugmAâµ 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 AnugmAâµ 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.
Asunto(s)
Aspergillus fumigatus/enzimología , Aspergillus nidulans/metabolismo , Pared Celular/enzimología , Hifa/metabolismo , Transferasas Intramoleculares/genética , Sustitución de Aminoácidos , Antifúngicos/farmacología , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/crecimiento & desarrollo , Bencenosulfonatos/farmacología , Caspofungina , Equinocandinas/farmacología , Polisacáridos Fúngicos/metabolismo , Hifa/crecimiento & desarrollo , Transferasas Intramoleculares/biosíntesis , Itraconazol/farmacología , Lipopéptidos , Pruebas de Sensibilidad Microbiana , Transporte de ProteínasRESUMEN
Pyranose-furanose mutases are essential enzymes in the life cycle of a number of microorganisms, but are absent in mammalian systems, and hence represent novel targets for drug development. To date, all such mutases show preferential recognition of a single substrate (e.g., UDP-Gal). We report here the detailed structural characterization of the first bifunctional pyranose-furanose mutase, which recognizes both UDP-Gal and UDP-GalNAc. The enzyme under investigation (cjUNGM) is involved in the biosynthesis of capsular polysaccharides (CPSs) in Campylobacter jejuni 11168. These CPSs are known virulence factors that are required for adhesion and invasion of human epithelial cells. Using a combination of UV/visible spectroscopy, X-ray crystallography, saturation transfer difference NMR spectroscopy, molecular dynamics and CORCEMA-ST calculations, we have characterized the binding of the enzyme to both UDP-Galp and UDP-GalpNAc, and compared these interactions with those of a homologous monofunctional mutase enzyme from E. coli (ecUGM). These studies reveal that two arginines in cjUNGM, Arg59 and Arg168, play critical roles in the catalytic mechanism of the enzyme and in controlling its specificity to ultimately lead to a GalfNAc-containing CPS. In ecUGM, these arginines are replaced with histidine and lysine, respectively, and this results in an enzyme that is selective for UDP-Gal. We propose that these changes in amino acids allow C. jejuni 11168 to produce suitable quantities of the sugar nucleotide substrate required for the assembly of a CPS containing GalfNAc, which is essential for viability.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Campylobacter/terapia , Campylobacter jejuni/enzimología , Transferasas Intramoleculares/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Biocatálisis , Infecciones por Campylobacter/metabolismo , Infecciones por Campylobacter/patología , Cristalografía por Rayos X , Escherichia coli/enzimología , Humanos , Transferasas Intramoleculares/química , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato N-Acetilgalactosamina/química , Uridina Difosfato N-Acetilgalactosamina/metabolismoRESUMEN
The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD(+) and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s(-1), respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.
Asunto(s)
Aspergillus nidulans/enzimología , Proteínas Fúngicas/metabolismo , UDPglucosa 4-Epimerasa/metabolismo , Secuencia de Aminoácidos , Aspergillus nidulans/genética , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , NAD/química , NAD/metabolismo , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , UDPglucosa 4-Epimerasa/química , UDPglucosa 4-Epimerasa/genética , Uridina Difosfato Glucosa/química , Uridina Difosfato Glucosa/metabolismoRESUMEN
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.