RESUMO
Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.
Assuntos
Concanavalina A/química , Concanavalina A/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Precursores de Proteínas/metabolismo , Sítios de Ligação , Canavalia/enzimologia , Domínio Catalítico , Dicroísmo Circular , Concanavalina A/genética , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Concentração de Íons de Hidrogênio , Metilmanosídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SoluçõesRESUMO
The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
Assuntos
Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Nucleossomos/metabolismo , Humanos , Modelos MolecularesRESUMO
Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.
Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Cristalografia por Raios X , DNA/química , Dimerização , Ligantes , Simulação de Dinâmica Molecular , Mutação , Poli A/química , Água/químicaRESUMO
Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.
Assuntos
Arabidopsis/enzimologia , Clitoria/enzimologia , Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Arabidopsis/genética , Evolução Biológica , Catálise , Domínio Catalítico , Clitoria/genética , Cristalografia por Raios X , Ciclização , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Ligases/química , Ligases/genética , Modelos Estruturais , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismoRESUMO
Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower, Helianthus annuus. The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.
Assuntos
Cisteína Endopeptidases/metabolismo , Helianthus/enzimologia , Helianthus/metabolismo , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Ribossomos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Conformação ProteicaRESUMO
The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES: Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.
Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Nucleossomos/química , Mapeamento de Interação de Proteínas/métodos , Animais , Artefatos , Western Blotting , Escherichia coli , Células HEK293 , Células HeLa , Histona Desacetilase 1/química , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas , Coelhos , Proteínas Recombinantes de Fusão/química , ReticulócitosRESUMO
The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.
Assuntos
Histona Desacetilases/química , Nucleossomos/química , Subunidades Proteicas/química , Proteínas Repressoras/química , Proteína 4 de Ligação ao Retinoblastoma/química , Proteína 7 de Ligação ao Retinoblastoma/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , Reagentes de Ligações Cruzadas/química , Expressão Gênica , Células HEK293 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Nucleossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/genética , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Alinhamento de Sequência , Termodinâmica , Transativadores , Transcrição GênicaRESUMO
The combination of stable isotope labelling with direct infusion ion mobility mass spectrometry (IM-MS) enabled qualitative and quantitative monitoring of biocatalytic reactions with reduced analysis times, enhanced sensitivity and µL-level assay volumes. The new approach was demonstrated by applying to both lipase and monooxygenase enzymes, including multi-substrate screening.
Assuntos
Biocatálise , Lipase/metabolismo , Espectrometria de Massas/métodos , Aminas/química , Ésteres , Pseudomonas stutzeri/enzimologia , Fatores de TempoRESUMO
Locus PA4043 in the genome of Pseudomonas aeruginosa PAO1 has been annotated as coding for a farnesyl pyrophosphate synthase (FPPS). This open reading frame was cloned and expressed recombinantly in Escherichia coli. The dimeric enzyme shows farnesyl pyrophosphate synthase activity and is strongly inhibited by ibandronate and zoledronate, drugs that are presently in clinical use. The structures of the unliganded enzyme and complexes with the substrate geranyl diphosphate (GPP), the inhibitor ibandronate and two compounds obtained from a differential scanning fluorimetry-based screen of a fragment library were determined by X-ray crystallography to resolutions of better than 2.0â Å. The enzyme shows the typical α-helical fold of farnesyl pyrophosphate synthases. The substrate GPP binds in the S1 substrate site in an open conformation of the enzyme. In the enzyme-ibandronate complex three inhibitor molecules are bound in the active site of the enzyme. One inhibitor molecule occupies the allylic substrate site (S1) of each subunit, as observed in complexes of nitrogen-containing bisphosphonate inhibitors of farnesyl synthases from other species. Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site. The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site. This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes.
Assuntos
Proteínas de Bactérias/química , Difosfonatos/química , Inibidores Enzimáticos/química , Geraniltranstransferase/química , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Cristalografia por Raios X , Geraniltranstransferase/antagonistas & inibidores , Ácido Ibandrônico , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
Assuntos
Proteínas de Bactérias/química , Pseudomonas aeruginosa/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
BACKGROUND: A central tenet of structural biology is that related proteins of common function share structural similarity. This has key practical consequences for the derivation and analysis of protein structures, and is exploited by the process of "molecular sieving" whereby a common core is progressively distilled from a comparison of two or more protein structures. This paper reports a novel web server for "sieving" of protein structures, based on the multiple structural alignment program MUSTANG. METHODOLOGY/PRINCIPAL FINDINGS: "Sieved" models are generated from MUSTANG-generated multiple alignment and superpositions by iteratively filtering out noisy residue-residue correspondences, until the resultant correspondences in the models are optimally "superposable" under a threshold of RMSD. This residue-level sieving is also accompanied by iterative elimination of the poorly fitting structures from the input ensemble. Therefore, by varying the thresholds of RMSD and the cardinality of the ensemble, multiple sieved models are generated for a given multiple alignment and superposition from MUSTANG. To aid the identification of structurally conserved regions of functional importance in an ensemble of protein structures, Lesk-Hubbard graphs are generated, plotting the number of residue correspondences in a superposition as a function of its corresponding RMSD. The conserved "core" (or typically active site) shows a linear trend, which becomes exponential as divergent parts of the structure are included into the superposition. CONCLUSIONS: The application addresses two fundamental problems in structural biology: first, the identification of common substructures among structurally related proteins--an important problem in characterization and prediction of function; second, generation of sieved models with demonstrated uses in protein crystallographic structure determination using the technique of Molecular Replacement.
Assuntos
Biologia Computacional/métodos , Software , Homologia Estrutural de Proteína , Algoritmos , Cristalografia por Raios X , Proteínas/químicaRESUMO
BACKGROUND: The crystallographic determination of protein structures can be computationally demanding and for difficult cases can benefit from user-friendly interfaces to high-performance computing resources. Molecular replacement (MR) is a popular protein crystallographic technique that exploits the structural similarity between proteins that share some sequence similarity. But the need to trial permutations of search models, space group symmetries and other parameters makes MR time- and labour-intensive. However, MR calculations are embarrassingly parallel and thus ideally suited to distributed computing. In order to address this problem we have developed MrGrid, web-based software that allows multiple MR calculations to be executed across a grid of networked computers, allowing high-throughput MR. METHODOLOGY/PRINCIPAL FINDINGS: MrGrid is a portable web based application written in Java/JSP and Ruby, and taking advantage of Apple Xgrid technology. Designed to interface with a user defined Xgrid resource the package manages the distribution of multiple MR runs to the available nodes on the Xgrid. We evaluated MrGrid using 10 different protein test cases on a network of 13 computers, and achieved an average speed up factor of 5.69. CONCLUSIONS: MrGrid enables the user to retrieve and manage the results of tens to hundreds of MR calculations quickly and via a single web interface, as well as broadening the range of strategies that can be attempted. This high-throughput approach allows parameter sweeps to be performed in parallel, improving the chances of MR success.
Assuntos
Biologia Computacional/métodos , Software , Homologia Estrutural de Proteína , Cristalografia por Raios X , Internet , Linguagens de Programação , Proteínas/químicaRESUMO
Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.
Assuntos
Proteínas de Bactérias/química , Eucariotos/enzimologia , Legionella pneumophila/enzimologia , Estrutura Terciária de Proteína , Pirofosfatases/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Alinhamento de SequênciaRESUMO
Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II alpha-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from D-haloalkanoic acids and in some cases also the L-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I alpha-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2(1)), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 A, alpha = 90, beta = 93.7, gamma = 90 degrees , and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary.
Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Pseudomonas putida/enzimologia , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X/métodos , Hidrolases/isolamento & purificação , EstereoisomerismoRESUMO
Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I alpha-haloacid dehalogenase that can process both L- and D-substrates. The structure shows that the DehI monomer consists of two domains of approximately 130 amino acids that have approximately 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both L- and D-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to D-only enzymes. These details will assist with future bioengineering of dehalogenases.
Assuntos
Proteínas de Bactérias/química , Hidrolases/química , Pseudomonas putida/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estereoisomerismo , Especificidade por SubstratoRESUMO
BACKGROUND: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. RESULTS: As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 A resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. CONCLUSION: Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína Adaptadora GRB7/antagonistas & inibidores , Proteína Adaptadora GRB7/química , Neoplasias/patologia , Peptídeos Cíclicos/farmacologia , Domínios de Homologia de src , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a "lock down" mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.
Assuntos
Proteínas de Bactérias/química , Burkholderia cepacia/enzimologia , Hidrolases/química , Modelos Moleculares , Sequência de Aminoácidos , Sítios de Ligação , Hidrocarbonetos Clorados , Hidrólise , Ligantes , Dados de Sequência Molecular , Propionatos/química , Conformação Proteica , Especificidade por Substrato , Água/químicaRESUMO
DehIVa is one of two dehalogenases produced by the soil- and water-borne bacterium Burkholderia cepacia MBA4. It acts to break down short-chain halogenated aliphatic acids through a nucleophilic attack and subsequent hydrolysis of an enzyme-substrate intermediate to remove the halide ions from L-enantiomers substituted at the C2 position (e.g L-2-monochloropropionic acid). Dehalogenases are an important group of enzymes that are responsible for breaking down a diverse range of halogenated environmental pollutants. The dhlIVa gene coding for DehIVa was expressed in Escherichia coli and the protein was purified and crystallized using the hanging-drop method. Crystals grown in PEG 4000 and ammonium sulfate diffracted to 3.1 A. The crystals had a primitive hexagonal unit cell, with unit-cell parameters a = b = 104.2, c = 135.8 A, alpha = beta = 90, gamma = 120 degrees. Determining this structure will provide valuable insights into the characterization of the catalytic mechanisms of this group of enzymes.