RESUMO
The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an â¼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.
Assuntos
Proteínas de Bactérias , Mycobacterium smegmatis , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Modelos Moleculares , Sequência de Aminoácidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13-19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate Tm values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.
Assuntos
Burkholderia , Mycobacterium avium subsp. paratuberculosis , Difosfatos , Cristalografia por Raios XRESUMO
Inorganic pyrophosphate (PPi) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PPi must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PPi into two orthophosphates (Pi), preventing the toxic accumulation of the PPi byproduct in cells and making Pi available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0â Å resolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) ß-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg2+ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.
Assuntos
Legionella pneumophila , Doença dos Legionários , Humanos , Legionella pneumophila/genética , Pirofosfatase Inorgânica/genética , Cristalografia por Raios X , Doença dos Legionários/genética , Doença dos Legionários/microbiologiaRESUMO
Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Influenza Humana , Humanos , Hemaglutininas , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Proteínas Virais , Anticorpos NeutralizantesRESUMO
Plasmepsin X (PMX) is an essential aspartyl protease controlling malaria parasite egress and invasion of erythrocytes, development of functional liver merozoites (prophylactic activity), and blocking transmission to mosquitoes, making it a potential multistage drug target. We report the optimization of an aspartyl protease binding scaffold and the discovery of potent, orally active PMX inhibitors with in vivo antimalarial efficacy. Incorporation of safety evaluation early in the characterization of PMX inhibitors precluded compounds with a long human half-life (t1/2) to be developed. Optimization focused on improving the off-target safety profile led to the identification of UCB7362 that had an improved in vitro and in vivo safety profile but a shorter predicted human t1/2. UCB7362 is estimated to achieve 9â¯logâ¯10 unit reduction in asexual blood-stage parasites with once-daily dosing of 50 mg for 7 days. This work demonstrates the potential to deliver PMX inhibitors with in vivo efficacy to treat malaria.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária , Animais , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Plasmodium falciparum/metabolismo , Ácido Aspártico Endopeptidases , Malária/tratamento farmacológicoRESUMO
BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.
Assuntos
Proteínas de Bactérias , Bactérias Gram-Negativas , Leishmania major , Peptidilprolil Isomerase , Proteínas de Protozoários , Proteínas de Bactérias/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Macrófagos/metabolismo , Neisseria meningitidis , Peptidilprolil Isomerase/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Proteínas RecombinantesRESUMO
Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.
Assuntos
Ácido Aspártico Endopeptidases , Precursores Enzimáticos , Plasmodium falciparum , Proteínas de Protozoários , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Precursores Enzimáticos/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/químicaRESUMO
Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Flavobacteriaceae/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Flavobacteriaceae/efeitos dos fármacos , Genoma Bacteriano/genéticaRESUMO
Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.
Assuntos
Descoberta de Drogas , Naegleria fowleri/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Simulação de Dinâmica Molecular , Naegleria fowleri/genética , Fosfoglicerato Mutase/antagonistas & inibidores , Fosfoglicerato Mutase/química , Fosfoglicerato Mutase/metabolismo , Estrutura Quaternária de Proteína , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteoma , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0â Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.
Assuntos
Oxirredutases do Álcool/química , Proteínas de Bactérias/química , Rickettsia felis/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
We identified and explored the structure-activity-relationship (SAR) of an adamantane carboxamide chemical series of Ebola virus (EBOV) inhibitors. Selected analogs exhibited half-maximal inhibitory concentrations (EC50 values) of â¼10-15 nM in vesicular stomatitis virus (VSV) pseudotyped EBOV (pEBOV) infectivity assays, low hundred nanomolar EC50 activity against wild type EBOV, aqueous solubility >20 mg/mL, and attractive metabolic stability in human and nonhuman liver microsomes. X-ray cocrystallographic characterizations of a lead compound with the EBOV glycoprotein (GP) established the EBOV GP as a target for direct compound inhibitory activity and further provided relevant structural models that may assist in identifying optimized therapeutic candidates.
RESUMO
Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.
Assuntos
Chlamydia trachomatis/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Neisseria gonorrhoeae/enzimologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified. The Seattle Structural Genomics Center for Infectious Disease entered 342 of these candidate essential genes into our pipeline for structure determination, in which 306 were successfully cloned into expression vectors, 192 were detectably expressed, 165 screened as soluble, 121 were purified, 52 crystalized, 30 provided diffraction data, and 29 structures were deposited in the Protein Data Bank. Here, we report these structures, compare them with human orthologs where applicable, and discuss their potential as drug targets for antibiotic development against A. baumannii.
Assuntos
Acinetobacter baumannii/química , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Coproporfirinogênio Oxidase/química , Coproporfirinogênio Oxidase/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Metionina tRNA Ligase/química , Metionina tRNA Ligase/metabolismo , Modelos Moleculares , Conformação Proteica , Uroporfirinogênio Descarboxilase/química , Uroporfirinogênio Descarboxilase/metabolismoRESUMO
Thiamine monophosphate kinase (ThiL) catalyzes the last step of thiamine pyrophosphate (TPP) synthesis, the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to thiamine pyrophosphate. We solved the structure of ThiL from the human pathogen A. baumanii in complex with a pair of substrates TMP and a non-hydrolyzable adenosine triphosphate analog, and in complex with a pair of products TPP and adenosine diphosphate. High resolution of the data and anomalous diffraction allows for a detailed description of the binding mode of substrates and products, and their metal environment. The structures further support a previously proposed in-line attack reaction mechanism and show a distinct variability of metal content of the active site.
Assuntos
Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Cristalização , Tiamina Pirofosfato/metabolismoRESUMO
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
Assuntos
Criptosporidiose , Cryptosporidium parvum/enzimologia , Inibidores Enzimáticos/farmacologia , Lisina-tRNA Ligase/antagonistas & inibidores , Malária Falciparum , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Criptosporidiose/tratamento farmacológico , Criptosporidiose/enzimologia , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Humanos , Lisina-tRNA Ligase/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Falciparum/enzimologia , Camundongos SCID , Proteínas de Protozoários/metabolismoRESUMO
Crystal structure determination requires solving the phase problem. This can be accomplished using ab initio direct methods for small molecules and macromolecules at resolutions higher than 1.2â Å, whereas macromolecular structure determination at lower resolution requires either molecular replacement using a homologous structure or experimental phases using a derivative such as covalent labeling (for example selenomethionine or mercury derivatization) or heavy-atom soaking (for example iodide ions). Here, a case is presented in which crystals were obtained from a 30.8â kDa protein sample and yielded a 1.6â Å resolution data set with a unit cell that could accommodate approximately 8â kDa of protein. Thus, it was unclear what had been crystallized. Molecular replacement with pieces of homologous proteins and attempts at iodide ion soaking failed to yield a solution. The crystals could not be reproduced. Sequence-independent molecular replacement using the structures available in the Protein Data Bank also failed to yield a solution. Ultimately, ab initio structure solution proved successful using the program ARCIMBOLDO, which identified two α-helical elements and yielded interpretable maps. The structure was the C-terminal dimerization domain of the intended target from Mycobacterium smegmatis. This structure is presented as a user-friendly test case in which an unknown protein fragment could be determined using ARCIMBOLDO.
Assuntos
Proteínas de Bactérias/química , GTP Fosfo-Hidrolases/química , Guanosina Difosfato/química , Mycobacterium smegmatis/química , Fragmentos de Peptídeos/química , Software , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Bases de Dados de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The bacterium Burkholderia phymatum is a promiscuous symbiotic nitrogen-fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. Other Burkholderia species are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase from B. phymatum was produced and crystallized, and a 1.95â Å resolution structure is reported. This enzyme shares less than 33% sequence identity with other homologs of known structure. There are two classes of chorismate mutase: AroQ and AroH. The bacterial subclass AroQγ has reported roles in virulence. Chorismate mutase from B. phymatum has the prototypical AroQγ topology and retains the characteristic chorismate mutase active site. This suggests that substrate-based chorismate mutase inhibitors will not be specific and are likely to affect beneficial bacteria such as B. phymatum.
Assuntos
Burkholderiaceae/enzimologia , Corismato Mutase/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Homologia de SequênciaRESUMO
Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity at the low pH environment of lysosomes where it is typically found in higher eukaryotes. Interestingly, DNase II has also been identified in a few genera of bacteria and is believed to have arisen via horizontal transfer. Here, we demonstrate that recombinant Burkholderia thailandensis DNase II is highly active at low pH in the absence of divalent metal ions, similar to eukaryotic DNase II. The crystal structure of B. thailandensis DNase II shows a dimeric quaternary structure which appears capable of binding double-stranded DNA. Each monomer of B. thailandensis DNase II exhibits a similar overall fold as phospholipase D (PLD), phosphatidylserine synthase (PSS) and tyrosyl-DNA phosphodiesterase (TDP), and conserved catalytic residues imply a similar mechanism. The structural and biochemical data presented here provide insights into the atomic structure and catalytic mechanism of DNase II.
Assuntos
Proteínas de Bactérias/química , Burkholderia/enzimologia , Endodesoxirribonucleases/química , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cobre/farmacologia , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/metabolismo , Células Eucarióticas/enzimologia , Evolução Molecular , Transferência Genética Horizontal , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Acoplamento Molecular , Filogenia , Células Procarióticas/enzimologia , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Aminoacyl-tRNA synthetases (aaRSs) charge tRNAs with their cognate amino acid, an essential precursor step to loading of charged tRNAs onto the ribosome and addition of the amino acid to the growing polypeptide chain during protein synthesis. Because of this important biological function, aminoacyl-tRNA synthetases have been the focus of anti-infective drug development efforts and two aaRS inhibitors have been approved as drugs. Several researchers in the scientific community requested aminoacyl-tRNA synthetases to be targeted in the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure determination pipeline. Here we investigate thirty-one aminoacyl-tRNA synthetases from infectious disease organisms by co-crystallization in the presence of their cognate amino acid, ATP, and/or inhibitors. Crystal structures were determined for a CysRS from Borrelia burgdorferi bound to AMP, GluRS from Borrelia burgdorferi and Burkholderia thailandensis bound to glutamic acid, a TrpRS from the eukaryotic pathogen Encephalitozoon cuniculi bound to tryptophan, a HisRS from Burkholderia thailandensis bound to histidine, and a LysRS from Burkholderia thailandensis bound to lysine. Thus, the presence of ligands may promote aaRS crystallization and structure determination. Comparison with homologous structures shows conformational flexibility that appears to be a recurring theme with this enzyme class.
Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Borrelia burgdorferi/enzimologia , Burkholderia/enzimologia , Encephalitozoon cuniculi/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Doenças Transmissíveis/microbiologia , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.