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1.
J Biol Chem ; 292(50): 20461-20471, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-28972175

RESUMEN

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn24 and Asp39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.


Asunto(s)
Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Neisseria meningitidis/enzimología , Tioléster Hidrolasas/metabolismo , Acetilcoenzima A/química , Acilcoenzima A/química , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Guanosina Difosfato/química , Mutación , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Tioléster Hidrolasas/química , Tioléster Hidrolasas/genética , Difracción de Rayos X
2.
J Biol Chem ; 291(4): 1866-1876, 2016 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-26538563

RESUMEN

PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn(37), Asp(52), and Thr(68) are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr(38)-Tyr(39). This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.


Asunto(s)
Acetilcoenzima A/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Coenzima A/metabolismo , Streptococcus pneumoniae/enzimología , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismo , Acetilcoenzima A/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico , Coenzima A/química , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Estructura Terciaria de Proteína , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética , Especificidad por Sustrato , Tioléster Hidrolasas/genética
3.
Artículo en Inglés | MEDLINE | ID: mdl-24192375

RESUMEN

Neisseria meningitidis is the causative microorganism of many human diseases, including bacterial meningitis; together with Streptococcus pneumoniae, it accounts for approximately 80% of bacterial meningitis infections. The emergence of antibiotic-resistant strains of N. meningitidis has created a strong urgency for the development of new therapeutics, and the high-resolution structural elucidation of enzymes involved in cell metabolism represents a platform for drug development. Acetyl-CoA hydrolase is involved in multiple functions in the bacterial cell, including membrane synthesis, fatty-acid and lipid metabolism, gene regulation and signal transduction. Here, the first recombinant protein expression, purification and crystallization of a hexameric acetyl-CoA hydrolase from N. meningitidis are reported. This protein was crystallized using the hanging-drop vapour-diffusion technique at pH 8.5 and 290 K using ammonium phosphate as a precipitant. Optimized crystals diffracted to 2.0 Šresolution at the Australian Synchrotron and belonged to space group P2(1)3 (unit-cell parameters a = b = c = 152.2 Å), with four molecules in the asymmetric unit.


Asunto(s)
Acetil-CoA Hidrolasa/química , Acetil-CoA Hidrolasa/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Neisseria meningitidis/enzimología , Cristalización , Electroforesis en Gel de Poliacrilamida , Humanos , Difracción de Rayos X
4.
Cells ; 12(14)2023 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-37508494

RESUMEN

Polyamines are simple yet critical molecules with diverse roles in numerous pathogenic and non-pathogenic organisms. Regulating polyamine concentrations affects the transcription and translation of genes and proteins important for cell growth, stress, and toxicity. One way polyamine concentrations are maintained within the cell is via spermidine/spermine N-acetyltransferases (SSATs) that acetylate intracellular polyamines so they can be exported. The bacterial SpeG enzyme is an SSAT that exhibits a unique dodecameric structure and allosteric site compared to other SSATs that have been previously characterized. While its overall 3D structure is conserved, its presence and role in different bacterial pathogens are inconsistent. For example, not all bacteria have speG encoded in their genomes; in some bacteria, the speG gene is present but has become silenced, and in other bacteria, it has been acquired on mobile genetic elements. The latter is the case for methicillin-resistant Staphylococcus aureus (MRSA) USA300, where it appears to aid pathogenesis. To gain a greater understanding of the structure/function relationship of SpeG from the MRSA USA300 strain (SaSpeG), we determined its X-ray crystal structure in the presence and absence of spermine. Additionally, we showed the oligomeric state of SaSpeG is dynamic, and its homogeneity is affected by polyamines and AcCoA. Enzyme kinetic assays showed that pre-incubation with polyamines significantly affected the positive cooperativity toward spermine and spermidine and the catalytic efficiency of the enzyme. Furthermore, we showed bacterial SpeG enzymes do not have equivalent capabilities to acetylate aminopropyl versus aminbutyl ends of spermidine. Overall, this study provides new insight that will assist in understanding the SpeG enzyme and its role in pathogenic and non-pathogenic bacteria at a molecular level.


Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Espermidina , Espermidina/metabolismo , Espermina/metabolismo , Poliaminas/metabolismo , Acetiltransferasas/metabolismo
5.
J Virol Methods ; 215-216: 1-8, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25698466

RESUMEN

Structural insights into the biology of viruses such as beak and feather disease virus (BFDV) which do not replicate in cell cultures are increasingly reliant on recombinant methods for protein production and purification. Development of efficient methods for homogenous production of BFDV capsid protein is also essential for vaccine development and diagnostic purposes. In this study, two different plasmids (pMCSG21 and pMCSG24), three homologous BFDV capsid proteins, and two unique expression media (auto-induction and IPTG-induced expression) were trialled for over-expression of the BFDV in Escherichia coli. Over-expression was observed for all three recombinant targets of BFDV capsid protein using E. coli BL21 (DE3) Rosetta 2 cell lines under IPTG induction. These proteins could be purified using an optimized, two-step purification process using a buffer containing 20mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 500 mM NaCl and supplemented with 200 mM L-arginine at pH 10.5, to yield a soluble and stable protein of greater than 95% purity. The final concentration of purified protein was approximately fourteen-to-eighteen fold greater than that reported previously. Initial crystallization and X-ray diffraction confirm that the protein is structured in a manner consistent with icosahedral symmetry. Antigenicity of recombinant Cap was confirmed by immunoassay, verifying its validity for use in continued experimentation as a potential DNA vaccine, a reagent in diagnostic assays, and purified concentrated protein for structural and functional biology.


Asunto(s)
Proteínas de la Cápside/aislamiento & purificación , Circovirus/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Medios de Cultivo/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos , Plásmidos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 2): 211-4, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24637759

RESUMEN

Staphylococcus aureus is a prevalent microorganism that is capable of causing a wide range of infections and diseases. Several strains of this bacterial species have developed antibiotic resistance to methicillin and vancomycin, and higher death rates are still being reported each year owing to multidrug-resistant strains. Certain GCN5-related N-acetyltransferases (GNATs) exhibit a broad substrate range, including aminoglycosides, histones, other proteins and serotonin, and have been implicated in antibiotic drug resistance. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of a GNAT from S. aureus (SaNAT) are reported. SaNAT was recombinantly expressed and crystallized by the hanging-drop vapour-diffusion method at 296 K, and the crystals diffracted to 1.7 Å resolution on the MX2 beamline at the Australian Synchrotron. The crystals belonged to space group P43212, with unit-cell parameters a = b = 84.86, c = 49.06 Å, α = ß = γ = 90°. A single molecule is likely to be present in the asymmetric unit. A full structural and functional analysis is currently being undertaken to provide novel insights into the protein function, which in turn may provide a basis for drug design.


Asunto(s)
Acetiltransferasas/química , Cristalografía por Rayos X/métodos , Staphylococcus aureus/enzimología , Acetiltransferasas/genética , Acetiltransferasas/aislamiento & purificación , Clonación Molecular , Cristalización , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Reacción en Cadena de la Polimerasa , Conformación Proteica
7.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 2): 244-7, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24637766

RESUMEN

Staphylococcus aureus is the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) from S. aureus subsp. aureus Mu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization of SaPaaI thioesterase from S. aureus are reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24% PEG 4000, 100 mM sodium citrate pH 6.5, 12% 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 Å resolution at the Australian Synchrotron and belonged to space group P12(1)1, with unit-cell parameters a = 44.05, b = 89.05, c = 60.74 Å, ß = 100.5°. Initial structure determination and refinement gave an R factor and R(free) of 17.3 and 22.0%, respectively, confirming a positive solution in obtaining phases using molecular replacement.


Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Esterasas/química , Staphylococcus aureus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Cristalización , Cartilla de ADN , Esterasas/genética , Esterasas/aislamiento & purificación , Conformación Proteica
8.
PLoS One ; 9(8): e102348, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25118709

RESUMEN

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic ß-bulge splayed at the ß-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.


Asunto(s)
Acetiltransferasas/química , Proteínas Bacterianas/química , Staphylococcus aureus/enzimología , Acetiltransferasas/aislamiento & purificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia
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