Human 3-hydroxyacyl-ACP dehydratase

Target ID:

HTD2A

Deposition Date:

Friday 21st August 2009

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

3IR3

Authors:

Ugochukwu, E.; Cocking, R.; Burgess-Brown, N.; Pilka, E.; Muniz, J.; Krojer, T.; Chaikuad, A.; Gileadi, O.; Bountra, C.; Arrowsmith, C. H.; Weigelt, J.; Edwards, A.; Kavanagh, K. L.; Oppermann, U.

Site:

Oxford

3IR3

tabs

Structure Details

In higher eukaryotes, the majority of fatty acids are synthesized by the cytosolic type I fatty acid synthase (FAS). FAS I is a large multi-domain protein with a total MW of 540 kDa where all seven catalytic domains are encoded on a single polypeptide and the functional unit is dimeric. More recently a mitochondrial fatty acid synthesis pathway has been described that is homologous to the bacterial FAS II system. In the FAS II system the catalytic steps are catalyzed by individual soluble proteins. In both cases the substrate is shuttled between active sites covalently attached through a thioester linkage to a phosphopantetheinyl arm on an acyl carrier protein (ACP). Human 3-hydroxyacyl-ACP dehydratase (HTD2) is a component of the mitochondrial FAS II system and a functional homologue of bacterial FabZ.

Fatty acids are synthesized de novo by the sequential addition of 2 carbon units to a growing acyl chain. The initial step in the elongation, catalyzed by ketoacyl-ACP synthase (KS), is the condensation of malonyl-ACP with acetyl-ACP (or acyl-ACP) to form acetoacetyl-ACP (or 3-ketoacyl-ACP) with the concomitant release of CO2. The ketone moiety is then reduced by a NADPH-dependent ketoacyl-ACP reductase (KR) resulting in 3-hydroxyacyl-ACP. This is dehydrated by 3-hydroxyacyl-ACP dehydratase (DH, HTD2) forming a trans-2-enoyl-ACP that is subsequently reduced by enoyl-ACP reductase (ER) to a fully saturated acyl-ACP. This cycle is repeated until the growing acyl chain is hydrolyzed from the ACP by a thioesterase. The end product of the cytosolic FAS is the 16-carbon palmitic acid while the main product of the mitochondrial FAS appears to be octanoyl-ACP, which is required for lipoic acid synthesis. Lipoic acid is an essential cofactor in enzymes involved in aerobic metabolism such as the E2 subunit of the pyruvate dehydrogenase complex that produces acetyl-CoA.

Disruption of mitochondrial FAS leads to an inability of yeast to grow on non-fermentable carbon sources, indicating it is necessary for mitochondrial function. More recently mitochondrial FAS has been linked with RNase P and RNA processing. RNase P is a ribonuceoprotein that cleaves the leader sequence of tRNAs generating the mature 5′ ends. In the yeast Saccharomyces cerevisiae , deletion of genes in the mitochondrial FAS II pathway leads to a loss in RNase P activity. In vertebrates HTD2 is encoded on a bicistonic mRNA downstream of a protein subunit of RNase P. The connection between RNA processing and mitochondrial fatty acid synthesis is unclear and is a current area of active investigation.

Human HTD2 is highly expressed in heart and liver with lower expression in kidney, spleen, skeletal muscle and placenta and is located on chromosome 3 at locus 3p14.3.

Materials and Methods

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Coding DNA sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGCTGCCAGTGCTGACCCTG
CAGCACTTTCAGCATATGCACATCAAAGTT
GGAGACCGCGCTGAACTTAGGAGGGCCTTC
ACACAGACTGATGTGGCTACCTTCTCAGAA
TTAACAGGGGATGTCAATCCTTTGCATTTG
AATGAAGACTTTGCAAAACACACCAAGTTT
GGAAATACAATTGTACATGGAGTTTTGATC
AACGGACTTATCTCAGCTCTCCTAGGAACT
AAAATGCCAGGGCCAGGCTGTGTATTTCTT
TCCCAGGAAATTAGCTTTCCAGCCCCTTTA
TATATTGGAGAAGTTGTTTTAGCTTCTGCA
GAAGTGAAAAAGCTGAAGCGGTTCATTGCT
ATTATTGCAGTGTCATGTTCTGTAATAGAA
AGTAAAAAGACTGTTATGGAAGGCTGGGTT
AAAGTTATGGTTCCAGAAGCTTCCAAATCC
TGACAGTAAAGGTGGATACGGATCCGAA

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq(*)smLPVL
TLQHFQHMHIKVGDRAELRRAFTQTDVATF
SELTGDVNPLHLNEDFAKHTKFGNTIVHGV
LINGLISALLGTKMPGPGCVFLSQEISFPA
PLYIGEVVLASAEVKKLKRFIAIIAVSCSV
IESKKTVMEGWVKVMVPEASKS

Tags and additions: Tag sequence: N-terminal His-tag with a TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq(*)sm; Tag removed: yes

Host: BL21(DE3)-R3-pRARE2

Expression protocol: : The glycerol stock of host strain BL21(DE3)-R3-pRARE2 was used to inoculate 10 ml of TB (terrific Broth) supplemented with 50 µg/ml kanamycin and 35 g/ml chloramphenicol. This starter culture was grown overnight at 37°C and used to inoculate a 1 liter culture in TB supplemented with 50 µg/ml kanamycin only. The culture was grown at 37°C until the OD₆₀₀ reached ~3.0. After that the temperature was lowered to 18°C. Protein production was induced with 0.1 mM IPTG and recombinant HTD2A was expressed at that temperature overnight. The next day cells were harvested by centrifugation at 5000 rpm for 20 minutes then the supernatant was discarded and pellets re-suspended in 70ml of 2x lysis buffer. Stored at-80°C.

Cell extraction: 2x Lysis buffer: 100 mM K-phosphate, pH 7.5, 1 M NaCl, 20% glycerol 0.5 mM TCEP; Procedure: Frozen cells, previously re-suspended, were thawed, and supplemented with: TCEP, Benzonase and protease inhibitors. Cells were lysed by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI (polyethyleneimine), stirring for 30 minutes, then centrifugation for 30 minutes at 17,000RPM. The supernatant was then further clarified by filtration (Acrodisc filters, 0.2 µm)

Column 1: Ni-affinity, HisTrap Crude FF, 5 ml (GE Healthcare)

Column 1 Buffers: 2 x Lysis buffer: 100 mM K-phosphate, pH 7.5, 1 M NaCl, 20% glycerol 0.5 mM TCEP; Wash buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 30 mM imidazole, 10% glycerol 0.5 mM TCEP; Elution buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 300 mM imidazole, 10% glycerol 0.5 mM TCEP.

Column 1 Procedure: The cell extract was loaded on the column at 4 ml/minute on an AKTA-express system (GE Healthcare). The column was washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 4 ml/min. The eluted peak of A280 was automatically collected.

Column 2: Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml (GE Healthcare)

Column 2 Buffers: Gel Filtration Buffer - 20mM HEPES pH 7.5, 250mM NaCl, 5% glycerol and 0.5 mM TCEP

Column 2 Procedure: The eluted fraction from the Ni-affinity Histrap column was loaded on the gel filtration column in GF buffer at 0.80 ml/min. Eluted proteins were collected in 2-ml fractions and analyzed on SDS-PAGE.

Column 3: Ni-NTA qiagen

Column 3 Buffers: Wash buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 30 mM imidazole, 10% glycerol 0.5 mM TCEP; Elution buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 300 mM imidazole, 10% glycerol 0.5 mM TCEP.

Enzymatic treatment: The amount of TEV enzyme used, in mgs, was calculated by using a 1:20 TEV to protein ratio in mgs. It was left incubating overnight at 4°C. The following day ran protein sample through 1ml slurry Nickel in a 10mm gravity column. Collected this as flow-through. Washed column 3 times with 1 ml of wash buffer each time. Finally eluted in 10mls of elution buffer and collected this as eluant. Pooled wash fractions and buffer exchanged in GF buffer using PD10 column.

Mass spectrometry characterization: ESI-MS revealed that the protein had a mass of 16171.0 Da (Expected mass 16171.1)

Compound: none

Protein concentration: Protein was stored in 20 mM HEPES pH7.5, 250 mM NaCl and 5% Glycerol at -80°C. The protein was concentrated to 7.2 mg/ml using a centricon with a 5 KDa cut off. The protein concentration was determined spectrophotometrically using ε²⁸⁰= 8480.

Crystallization: Crystals were grown at 4°C by vapour diffusion in sitting drops mixing protein (7.2 mg/ml) and well solution containing 0.5% jeff2001; 1.1M Na (malonate); 0.1M HEPES pH 7.0 at a protein to precipitant ratio of 1:2.
Crystals were cryo-protected using Malonate 7.0 and flash cooled in liquid nitrogen.

Data Collection: Resolution (estimated): 2 Å; Resolution (scaled): 1.99 Å; X-ray source: Dmnd I04

References

Autio, K. J., A. J. Kastaniotis, et al. (2008). "An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing." FASEB J 22(2): 569-78.

Hiltunen, J. K., M. S. Schonauer, et al. (2009). "Mitochondrial fatty acid synthesis type II: more than just fatty acids." J Biol Chem 284(14): 9011-5.

Schonauer, M. S., A. J. Kastaniotis, et al. (2008). "Intersection of RNA processing and the type II fatty acid synthesis pathway in yeast mitochondria." Mol Cell Biol 28(21): 6646-57.

Witkowski, A., A. K. Joshi, et al. (2007). "Coupling of the de novo fatty acid biosynthesis and lipoylation pathways in mammalian mitochondria." J Biol Chem 282(19): 14178-85.