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Expression, purification and preliminary crystallographic analysis of Rv2247, the ? subunit of acyl-CoA carboxylase (ACCD6) from Mycobacterium tuberculosis.

ABSTRACT: Mycobacterium tuberculosis (Mtb) acyl-CoA carboxylase is involved in the biosynthesis of mycolic acids, which are a key component of the bacillus cell wall. The Mtb genome encodes six acyl-CoA carboxylase ? subunits (ACCD1-6), three of which (ACCD4-6) are essential for survival of the pathogen on minimal medium. Mtb ACCD6 has been expressed, purified and crystallized. The two forms of Mtb ACCD6 crystals belonged to space groups P4(1)2(1)2 and P2(1)2(1)2(1) and diffracted to 2.9 and 2.5 Å resolution, respectively, at a synchrotron-radiation source.

SUBMITTER: Niu C

PROVIDER: S-EPMC3232159 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Expression, purification and preliminary crystallographic analysis of Rv2247, the β subunit of acyl-CoA carboxylase (ACCD6) from Mycobacterium tuberculosis.

Niu Chunying C Yin Jiang J Cherney Maia M MM James Michael N G MN

Acta crystallographica. Section F, Structural biology and crystallization communications 20111130 Pt 12

Mycobacterium tuberculosis (Mtb) acyl-CoA carboxylase is involved in the biosynthesis of mycolic acids, which are a key component of the bacillus cell wall. The Mtb genome encodes six acyl-CoA carboxylase β subunits (ACCD1-6), three of which (ACCD4-6) are essential for survival of the pathogen on minimal medium. Mtb ACCD6 has been expressed, purified and crystallized. The two forms of Mtb ACCD6 crystals belonged to space groups P4(1)2(1)2 and P2(1)2(1)2(1) and diffracted to 2.9 and 2.5 Å resolut ...[more]

PMID: 22139186

Similar Datasets

Carboxyltransferase Subunit of the Acyl-CoA Carboxylase in Mycobacterium tuberculosis.

Project description:The Mycobacterium tuberculosis acyl-CoA carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I end-products by the FAS II complex to produce meromycolic acids. M. tuberculosis genome contains three biotin carboxylase subunits (AccA1-3) and 6 carboxyltransferase subunits (AccD1-6) of which AccD6 is located in a genetic locus that contains members of the FAS II complex. We found by microarray and quantitative real-time RT-PCR analysis that the transcripts of AccA3, AccD4, AccD5 and AccD6 are expressed at high levels during exponential growth phases of M. tuberculosis in vitro. Keywords: Time course, developmental stages

2006-10-07 | GSE5977 | GEO

Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor.

Project description:The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.

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Expression, purification and preliminary crystallographic analysis of Rv3002c, the regulatory subunit of acetolactate synthase (IlvH) from Mycobacterium tuberculosis.

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Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans.

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Crystallization and preliminary crystallographic analysis of DtsR1, a carboxyltransferase subunit of acetyl-CoA carboxylase from Corynebacterium glutamicum.

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| S-EPMC2330119 | biostudies-literature

Structure-based inhibitor design of AccD5, an essential acyl-CoA carboxylase carboxyltransferase domain of Mycobacterium tuberculosis.

Project description:Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelope of pathogenic mycobacteria. These unusually long fatty acids are essential for the survival, virulence, and antibiotic resistance of Mycobacterium tuberculosis. Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms such as Escherichia coli or humans that have only one or two ACCases, M. tuberculosis contains six ACCase carboxyltransferase domains, AccD1-6, whose specific roles in the pathogen are not well defined. Previous studies indicate that AccD4, AccD5, and AccD6 are important for cell envelope lipid biosynthesis and that its disruption leads to pathogen death. We have determined the 2.9-Angstroms crystal structure of AccD5, whose sequence, structure, and active site are highly conserved with respect to the carboxyltransferase domain of the Streptomyces coelicolor propionyl-CoA carboxylase. Contrary to the previous proposal that AccD4-5 accept long-chain acyl-CoAs as their substrates, both crystal structure and kinetic assay indicate that AccD5 prefers propionyl-CoA as its substrate and produces methylmalonyl-CoA, the substrate for the biosyntheses of multimethyl-branched fatty acids such as mycocerosic, phthioceranic, hydroxyphthioceranic, mycosanoic, and mycolipenic acids. Extensive in silico screening of National Cancer Institute compounds and the University of California, Irvine, ChemDB database resulted in the identification of one inhibitor with a K(i) of 13.1 microM. Our results pave the way toward understanding the biological roles of key ACCases that commit acyl-CoAs to the biosynthesis of cell envelope fatty acids, in addition to providing a target for structure-based development of antituberculosis therapeutics.

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Purification, crystallization and preliminary X-ray crystallographic studies of Rv3899c from Mycobacterium tuberculosis.

Project description:Rv3899c, a hypothetical protein from Mycobacterium tuberculosis that is conserved within the mycobacteria, is predicted to be secreted and has been found in culture filtrates. Here, Rv3899c has been cloned, expressed in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion method with PEG 3350 as a precipitant was used to crystallize the protein. N-terminal sequencing results showed that the amino-acid sequence of the crystallized protein began with GATAG, indicating that it is a fragment containing residues 184-410 of Rv3899c. Rv3899c184-410 crystals exhibited the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a=49.88, b=54.72, c=75.52?Å, ?=?=?=90°, and diffracted to a resolution of 1.90?Å.

| S-EPMC4304759 | biostudies-literature

Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis.

Project description:The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6122 or P6522, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

| S-EPMC4118811 | biostudies-literature

Expression, purification and preliminary crystallographic analysis of O-acetylhomoserine sulfhydrylase from Mycobacterium tuberculosis.

Project description:The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1 Å.

| S-EPMC3151138 | biostudies-literature

HisB from Mycobacterium tuberculosis: cloning, overexpression in Mycobacterium smegmatis, purification, crystallization and preliminary X-ray crystallographic analysis.

Project description:HisB, encoded by open reading frame Rv1601, possesses enzymatic activity as an imidazoleglycerol-phosphate dehydratase in the histidine-biosynthetic pathway of Mycobacterium tuberculosis. A recombinant form of HisB was crystallized in three crystal forms: crystals grown using 20% PEG 1500 as a precipitant belonged to either the cubic space group P432 or the tetragonal space group P4, while an orthorhombic crystal form belonging to space group P2(1)2(1)2 was obtained using 15% PEG 5000 and 10 mM MnCl(2) as precipitant. The structure of HisB in the orthorhombic crystal form was solved by the molecular-replacement method using the crystal structure of its Arabidopsis thaliana counterpart, which shares 47% sequence identity with Rv1601, as the search model.

| S-EPMC3212474 | biostudies-literature

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