Human peroxisomal acetyl-CoA acyl transferase 1

Target ID:

ACAA1A

Aliases:

ACAAPTHIOTHIO

Deposition Date:

Tuesday 26th September 2006

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2IIK

Authors:

E.PAPAGRIGORIOU,C.JOHANSSON,C.SMEE,K.KAVANAGH,A.C.W.PIKE,M.SUNDSTROM,J.WEIGELT,A.EDWARDS,C.H.ARROWSMITH,O.GILEADI,F.GORREC,C.UMEANO,F.VON DELFT,U.OPPERMANN,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2IIK

tabs

Structure Details

Thiolases (ketoacyl-CoA acyl transferases) are essential enzymes involved in fatty acid synthesis and catabolism, found both in pro- and eukaryotes.
In the latter, they are present in cytosolic, mitochondrial and peroxisomal subcellular compartments.

Two distinct classes (thiolase I and thiolase II) have been defined.
Thiolase I enzymes catalyze the last reaction step in the β-oxidation pathway of fatty acids resulting in one molecule each of acyl- CoA and acetyl-CoA,
which are funnelled into subsequent rounds of β-oxidation and are further oxidized.
Different forms of type I thiolases are known with distinct subcellular localization and different acyl-chain length specificities.
Human peroxisomal acetyl-CoA acyl transferase 1 (ACAA1) belongs to the type I thiolase group and acts preferentially on long- and medium-chain length acyl-CoAs.

Expression levels of ACAA1 in cultivated human hepatocytes are apparently not directly regulated by peroxisome proliferator activated-receptor alpha (PPAR a ) agonists,
although ACAA1 belongs to peroxisome proliferation-associated genes.
The human ACAA1 gene is highly expressed in liver, but also found in other metabolic highly active tissues such as CNS, muscle, heart and kidney. The human gene is localized to the short arm of chromosome 3 (3p23).

The type II thiolases are enzymes involved in fatty acid and lipid synthesis by catalyzing the condensation of two molecules of acyl-CoAs to form ketoacyl-CoA.

Despite the biological differences in these enzymes, the underlying reaction follows a reversible Claisen condensation mechanism in both cases.

Materials and Methods

ACAA1 - Human peroxisomal acetyl-CoA acyl transferase 1

PDB:2IIK

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC000635
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) , TEV-cleavable (*), N-terminal his6 tag
Host:Rosetta-R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmAPQASA ADVVVVHGRRTAICRAGRGGFKDTTPDEL LSAVMTAVLKDVNLRPEQLGDICVGNVLQ PGAGAIMARIAQFLSDIPETVPLSTVNRQ CSSGLQAVASIAGGIRNGSYDIGMACGVE SMSLADRGNPGNITSRLMEKEKARDCLIP MGITSENVAERFGISREKQDTFALASQQK AARAQSKGCFQAEIVPVTTTVHDDKGTKR SITVTQDEGIRPSTTMEGLAKLKPAFKKD GSTTAGNSSQVSDGAAAILLARRSKAEEL GLPILGVLRSYAVVGVPPDIMGIGPAYAI PVALQKAGLTVSDVDIFEINEAFASQAAY CVEKLRLPPEKVNPLGGAVALGHPLGCTG ARQVITLLNELKRRGKRAYGVVSMCIGTG MGAAAVFEYPGN
Vector:pNIC28-Bsa4

Growth

Medium:TB + 50 µg/ml Kanamycin + 34 µg/ml chloramp.
Antibiotics:
Procedure:2 x 1 liter TB in 2.5-L baffled flasks were inoculated with 10 ml overnight culture and grown at 37°C. The protein expression was induced with 0.5 mM IPTG at OD600= 3.2 at 18°C overnight. The cells were collected by centrifugation and frozen at -80°C.

Purification

Procedure
Lysis buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP.
Wash buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 50 mM imidazole, 0.5 mM TCEP.
Elution buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 350 mM imidazole, 0.5 mM TCEP.
Gel filtration buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP Column 1: Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gel filtration, Hiload 16/60 Superdex 200 prep grade, 120 ml (GE/ Amersham Biosciences)
The eluted fractions from the Ni-affinity Histrap column were loaded on the gel filtration column at 1.0 ml/min. Eluted proteins were collected in 2 ml fractions.

Concentration : The protein was concentrated in Amicon (5 K) to 25.1 mg/ml and the protein concentration determined spectrophotometrically using the predicted molar extinction coefficient 43935(M-1cm-1).

Extraction

Procedure
Frozen cell pellets were thawed at 37°C and resuspended in a total volume of 100 ml lysis buffer. The cells were disrupted by high pressure (20 kpsi) followed by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI , followed by centrifugation for 30 minutes at 40,000xg. The supernatant was further clarified by filtration (0.45 µm).
Concentration:
Ligand
MassSpec:Two peaks of 43849.5 and 43937.2 Da were detected for ACAA1A p001, which are 68.5 and 19.2 Da higher than the expected mass of 43918 Da for the his-tagged protein.
Crystallization:Crystals were grown by vapor diffusion at 20°C from a sitting drop consisting of 150 nl protein (25.1 mg/ml), containing 2 mM CaCl2 and 10 mM acetylcoenzyme A, and 150 nl well solution. The drop was equilibrated against well solution containing 25% PEG 3350, 0.24 M ammonium sulfateand 100 mM HEPES pH 7.5. The crystal was transferred to a cryoprotectant composed of 20% ethylene glycol before flash-cooling in liquid nitrogen .
NMR Spectroscopy:
Data Collection:
Data Processing: