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Title: Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

Abstract

The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Authors:
;  [1];  [1];  [2];  [2];  [2]; ;  [1];  [2];  [2]
  1. School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072 (Australia)
  2. (Australia)
Publication Date:
OSTI Identifier:
22356271
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 62; Journal Issue: Pt 2; Other Information: PMCID: PMC2150959; PMID: 16511283; PUBLISHER-ID: pu5122; OAI: oai:pubmedcentral.nih.gov:2150959; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CARBOXYLIC ACIDS; CRYSTAL STRUCTURE; CRYSTALLIZATION; CRYSTALS; DIFFUSION; MOLECULES; RESOLUTION; SPACE GROUPS; SYMMETRY; X-RAY SOURCES

Citation Formats

Serek, Robert, Forwood, Jade K., Hume, David A., Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland 4072, Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072, Martin, Jennifer L., Kobe, Bostjan, E-mail: b.kobe@uq.edu.au, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, and Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase. United Kingdom: N. p., 2006. Web. doi:10.1107/S1744309106000030.
Serek, Robert, Forwood, Jade K., Hume, David A., Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland 4072, Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072, Martin, Jennifer L., Kobe, Bostjan, E-mail: b.kobe@uq.edu.au, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, & Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072. Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase. United Kingdom. doi:10.1107/S1744309106000030.
Serek, Robert, Forwood, Jade K., Hume, David A., Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland 4072, Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072, Martin, Jennifer L., Kobe, Bostjan, E-mail: b.kobe@uq.edu.au, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, and Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072. Wed . "Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase". United Kingdom. doi:10.1107/S1744309106000030.
@article{osti_22356271,
title = {Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase},
author = {Serek, Robert and Forwood, Jade K. and Hume, David A. and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072 and Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, Queensland 4072 and Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072 and Martin, Jennifer L. and Kobe, Bostjan, E-mail: b.kobe@uq.edu.au and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072 and Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland 4072},
abstractNote = {The C-terminal domain of the mouse long-chain acyl-CoA thioesterase has been expressed in bacteria and crystallized by vapour diffusion. The crystals diffract to 2.4 Å resolution. The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 Å, γ = 120°). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 Å resolution using the laboratory X-ray source and are suitable for crystal structure determination.},
doi = {10.1107/S1744309106000030},
journal = {Acta Crystallographica. Section F},
number = Pt 2,
volume = 62,
place = {United Kingdom},
year = {Wed Feb 01 00:00:00 EST 2006},
month = {Wed Feb 01 00:00:00 EST 2006}
}
  • The cDNA for mouse long-chain acyl-CoA dehydrogenase (Acadl, gene symbol; LCAD, enzyme) was cloned and characterized. The cDNA was obtained by library screening and reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence showed a high degree of homology to both the rat and the human LCAD sequence. Northern analysis of multiple tissues using the mouse Acadl cDNA as a probe showed two bands in all tissues examined. We found a total of three distinct mRNAs for Acadl. These three mRNAs were encoded by a single gene that we mapped to mouse chromosome 1. The three transcripts differed inmore » the 3{prime} untranslated region due to use of alternative polyadenylation sites. Quantitative evaluation of a multitissue Northern blot showed a varied ratio of the larger transcript as compared with the smaller transcripts. 40 refs., 6 figs., 1 tab.« less
  • Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-{angstrom} resolution. The overall fold of the N-terminal {approx}400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an {alpha}-helical bundle that ismore » positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12{angstrom} and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial {beta}-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.« less
  • Medium-chain acyl-CoA, dehydrogenase (MCAD) is one of the three straight-chain length-specific dehydrogenases involved in the first step of fatty acid oxidation. Inherited defects of acyl-CoA dehydrogenases occur in humans, and MCAD deficiency is the most common. We have cloned the coding and 3{prime} untranslated sequence of mouse MCAD cDNA. The mouse MCAD cDNA coding region is 1263 bp long with a 3{prime} untranlsated region of 576 bp and encodes a 421-amino acid precursor protein. Comparing the nucleotide and deduced amino acid sequences of the mouse MCAD cDNA to rat and human MCAD cDNAs reveals considerable similarity between species. Amino acidmore » residues where substitutions result in human MCAD deficiency are conserved in the mouse. Amino acid residues involved in important enzymatic functions are also conserved.« less
  • Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the [beta]-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of clone dPCR-amplified MCAD cDNA from a G985 compound heterozygous patient, the authors identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patientmore » and her mother by a PCR-based assay. The mutation changes a conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, the authors suggest that the mutation destroys a salt bridge between arginine[sup 28] and glutamate[sup 86], thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. The results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases. 42 refs., 5 figs., 3 tabs.« less
  • Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4,more » for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids being trafficking into PC and PI. Collectively, these data support the conclusion that FATP2 has a dual function in the pathways linking the transport and activation of exogenous fatty acids. We discuss the differential roles of FATP2 and its role in both fatty acid transport and fatty acid activation in the context of lipid homeostasis.« less