Human 3-oxoacid CoA transferase

Target ID:

OXCTA

Aliases:

OXCTOXCT1SCOT

Deposition Date:

Monday 30th June 2008

Families:

Miscellaneous

Structure type:

protein-ligand

Download Coordinates:

3DLX

Authors:

Kathryn Kavanagh, Naeem Shafqat, Wyatt Yue, Sarah Picaud, James Murray, Elizabeth Maclean, Frank von Delft, Annette Roos, Aled Edwards, Cheryl Arrowsmith, Mats Wikstrom, Chas Bountra, Udo Oppermann

Site:

Oxford

3DLX

tabs

Structure Details

Ketone bodies (acetoacetate and D-hydroxybutyrate) are major sources of energy under situations of enhanced fatty acid beta oxidation such as starvation or in situations of ketoacidosis such as diabetes mellitus. For utilization of acetoacetate in extrahepatic tissues (such as heart, brain, skeletal muscle and kidney) the mitochondrial enzyme 3-oxoacid CoA transferase catalyzes the transfer of succinyl-CoA to acetoacetate to form acetoacetyl-CoA, which is further converted by thiolase to form 2 molecules of acetyl-CoA. This product is then funneled into the citric acid cycle.

Several mutations in the human OXCT gene have been identified as being causal for recurrent ketoacidosis with an underlying defect in succinyl-CoA:3-oxoacid transferase (SCOT) deficiency, a rare form of ketoacidosis.

Materials and Methods

Vector: pNIC-CH. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: C-terminal His-tag with TEV protease cleavage site (Tag sequence in lowercase)

MTKFYTDPVEAVKDIPDGATVLVGGFGLC
GIPENLIDALLKTGVKGLTAVSNNAGVDN
FGLGLLLRSKQIKRMVSSYVGENAEFERQ
YLSGELEVELTPQGTLAERIRAGGAGVPA
FYTPTGYGTLVQEGGSPIKYNKDGSVAIA
SKPREVREFNGQHFILEEAITGDFALVKA
WKADRAGNVIFRKSARNFNLPMCKAAETT
VVEVEEIVDIGAFAPEDIHIPQIYVHRLI
KGEKYEKRIERLSIRKEGDGEAKSAKPGD
DVRERIIKRAALEFEDGMYANLGIGIPLL
ASNFISPNITVHLQSENGVLGLGPYPRQH
EADADLINAGKETVTILPGASFFSSDESF
AMIRGGHVDLTMLGAMQVSKYGDLANWMI
PGKMVKGMGGAMDLVSSAKTKVVVTMEHS
AKGNAHKIMEKCTLPLTGKQCVNRIITEK
AVFDVDKKKGLTLIELWEGLTVDDVQKST
GCDFAVSPKLMPMQQIANAENLYFQshhh
hhhdykddddk

Host: E. coli strain BL21(DE3)-R3 pRARE2

Growth medium, induction protocol: 10 µl of a glycerol stock was inoculated into 5ml of TB medium (supplemented with 50 µg/ml Kanamycin, 34 µg/ml Chloramphenicol) and cultured at 37°C o/n in a shaking incubator (275 rpm). Next day 0.75 ml of o/n culture was used to inoculate 1 litre of TB medium (4x) and grown at 37°C with vigorous shaking (160 rpm) until the culture reaches an OD₆₀₀ of 1.4. Temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 0.5 mM, and further cultivated for 16 hrs. Cells were harvested by centrifugation at 6500 rpm for 10 min, and the cell pellet was stored at -20°C until further use.

Extraction buffer, extraction method: Lysis buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 5 mM Imidazole, Complete® protease inhibitors (Roche, 1 tbl/50 ml). Frozen cell pellets were thawed and resuspended in a total volume of 30-40 ml of lysis buffer, and disrupted by using Avestin C-5 microfluidizer, and a supernatant containing the target protein was obtained by centrifugation at 21,000 (rpm) for 45 minutes.

Column 1: Ni-Sepharose 6 Fast Flow

Buffers: Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole; Wash buffer: 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% Glycerol, 30 mM Imidazole; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole. Note: All the buffers contain 0.5mM TCEP.

Procedure: The column was packed with 2 ml of Ni-Sepharose 6 Fast Flow slurry and equilibrated with 15 ml of binding buffer. The supernatant was loaded onto the column and the column was washed with 20 ml of binding buffer and then 20 ml of washing buffer. The protein was eluted with 10 ml of elution buffer.

Column 2: SuperDex 200 16/60 HiLoad (GE/Amersham)

Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP.

Procedure: The eluted protein from the Ni-affinity column was loaded on the gel filtration column in GF buffer at 1.0 ml/min on an AKTA Purifier system. Eluted proteins were collected in 1 ml fractions.

Enzymatic treatment: TEV cleaved.

Column 3: Ni-NTA (TEV clean up)

Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP

Procedure: Total 10 mgs of protein was cleaved with 600 ug of TEV protease at 4°C for 48 hours.

TEV clean up: The TEV cleaved protein was applied to a 1 ml Ni-NTA column, already equilibrated with gel filtration buffer (10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP). The flow through form the column was collected. The eluate from the column was monitored by SDS gel analysis.

Sample Concentration: The OXCTA protein (in buffer; 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) was concentrated to 21 mg/ml using Vivaspin 10K concentrators and stored at -80°C.

Mass spec characterization: Corresponds to theoretical mass, as determined by ESI-TOF MS.

Crystallization: Crystals were grown by vapour diffusion in sitting drops at 20°C. Before setting up the experiment acetyl-CoA was added to the protein to a final concentration of 2mM. A sitting drop consisting of 100 nl protein and 300 nl well solution was equilibrated against well solution containing 0.20 M sodium chloride, 0.1 M tris pH 8.5 and 25% w/v polyethylene glycol 3350. Crystals were cryo protected in 20% glycerol and flash-cooled in liquid nitrogen.

Data Collection, Resolution: 2.2 Å , X-ray source: Synchrotron SLS-X10SA, single wavelength.