human isopentenyl-diphosphate delta isomerase 2

Target ID:

IDI2

Aliases:

IPPI2

Deposition Date:

Wednesday 25th April 2007

Families:

Diabetes

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2PNY

Authors:

J.R.WALKER,T.DAVIS,C.BUTLER-COLE,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2PNY

tabs

Structure Details

In 1976, the Sankyo Company in Japan discovered fungal metabolites with hypocholesterolemic activity in rats and dogs and that inhibition was competitive with respect to HMG-CoA substrates [2]. A few years later therapeutic effects were confirmed in human clinical trials [4]. Today, related blockbuster drugs such as Lipitor, Zocor, and Pravachol effectively manage lipidemia, a major risk factor of cardiovascular disease.

HMG-CoA is the first and rate-limit enzyme in the isoprenoid biosynthetic pathway. Its reaction product is then sequentially bis-phosphorylated, decarboxylated, and isomerized to produce the C5 pyrophosphate intermediates isopentenyl and dimethyl-allyl. These are polymerized to produce geranyl and farnesyl compounds, which are used as post-translational modification agents and as precursors of cholesterol (and therefore steroid) biosynthesis. Therapeutic drugs directed at other enzymes in this metabolic pathway have yet to be fully developed.

Unlike other enzymes in this pathway, there are two human paralogs of the isomerization step (isopentenyl diphosphate isomerase 1 and 2, IDI1 and IDI2), both catalytically competent, the first highly conserved, ubiquitously expressed and most likely responsible for housekeeping isomerase activity and the second expressed at high levels only in skeletal muscle but whose biological role is unclear [1].

We have solved the structure of human IDI2, revealing the conserved nudix fold and a deeply buried active site whose overall structure is similar to IDI1 [5,6]. Our structure contains a bacterially-derived pyrophosphate molecule in the canonical binding pocket of the active site. The substrate binding pocket is surround by various residues; a hydrogen bonding network composed of a chelated zinc and glutamate side chain likely protonates the substrate forming the carbocationic intermediate and an antarafacial serine deprotonates the adjacent methylene [3]. The conformation of the unique amino acids in the active site of IDI2 could provide insight into potential differences in transition states stabilized by these paralogs.

Materials and Methods

IDI2

PDB:2PNY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_150286
Entry Clone Source:OBS-BC017778.1
SGC Clone Accession:idi2.001.227:45A03
Tag:mgsshhhhhhssglvprgs
Host:BL21(DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMSDINLDWVDRRQLQRLEEMLIVVDENDKVIGADTKRNCHLNENIEKGLLHRAFSVVLFNTKNRILIQQRSDTKVTFPGYFTDSCSSHPLYNPAELEEKDAIGVRRAAQRRLQAELGIPGEQISPEDIVFMTIYHHKAKSDRIWGEHEICYLLLVRKNVTLNPDPSETKSILYLSQEELWELLEREARGEVKVTPWLRTIAERFLYRWWPHLDDVTPFVELHKIHRV
Vector:p28a-thrombin-lic

Growth

Medium:Terrific Broth (TB)
Antibiotics:
Procedure:Idi2 was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37ºC to an OD600 of 7.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.1 mM, and incubated overnight at 15ºC. The culture was centrifuged and the cell pellets were collected and stored at -80ºC.

Purification

Procedure
Column 1: His- Ni-NTA resin (Qiagen 30450)
Column 2: XK 16x65 column (GE Healthcare)
Lysed cells were diluted to 50 -100 mL final volume and imidazole added to a final concentration of 10 mM; this was then mixed with 2-3 mL of HisLink Protein Purification Resin (Promega V8821) per construct. The mixture was incubated with mixing for at least 20 minutes at 4 degC. The lysate was spun at 500xg for 3 minutes to pellet the HisLink resin. The lysate was carefully decanted off the resin, and then 50 mL of lysis buffer were added to wash the resin. The resin was allowed to settle for 5 minutes, then poured off and washed 3 more times with fresh lysis buffer. The washed resin was then loaded onto a gravity column, and then washed with a column volume of low imidazole buffer. Samples were eluted from the HisLink resin by exposure to 10-14 mL MAC elution buffer at a 1mL/min flow rate. An XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes at a flow rate of 1.5 mL/min. 10 mL of sample was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected. Peak fractions were analyzed for purity and pooled. A mixture of MgCl2, MnCl2, and CaCl2 (final concentration of each: 1mM) was added to purified protein, and then the mixture was concentrated using 15 mL concentrators with 10,000 MWCO (Amicon, UFC901024) to a final concentration of 10 mg/mL for crystallographic screening.

Extraction

Procedure
Cell pellets contained in bags (Beckman 369256) obtained from 4L culture were thawed by soaking in warm water. Each cell pellet was resuspended in 20 mL lysis buffer and then homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol is 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 6 minutes total sonication time per pellet.
Concentration:10 mg/mL
Ligand
MassSpec:
Crystallization:Diffraction quality crystals were grown using the following protocol: 2M sodium/potassium phosphate, pH 7.0 were mixed in a ratio of 0.5 microL protein+0.5 microL reservoir in sitting drop trays and placed at 18 degC. Resultant crystals were cryoprotected in 20% glycerol and frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

D.B. Clizbe, M.L. Owens, K.R. Masuda, J.E. Shackelford, S.K. Krisans, IDI2, a second isopentenyl diphosphate isomerase in mammals. J.Biol.Chem. 282 (2007) 6668-6676.
A. Endo, M. Kuroda, K. Tanzawa, Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity. FEBS Lett. 72 (1976) 323-326.
S. Lee, C.D. Poulter, Escherichia coli type I isopentenyl diphosphate isomerase: structural and catalytic roles for divalent metals. J.Am.Chem.Soc. 128 (2006) 11545-11550.
A. Yamamoto, H. Sudo, A. Endo, Therapeutic effects of ML-236B in primary hypercholesterolemia. Atherosclerosis 35 (1980) 259-266.
C. Zhang, L. Liu, H. Xu, Z. Wei, Y. Wang, Y. Lin, W. Gong, Crystal structures of human IPP isomerase: new insights into the catalytic mechanism. J.Mol.Biol. 366 (2007) 1437-1446.
W. Zheng, F. Sun, M. Bartlam, X. Li, R. Li, Z. Rao, The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis. J.Mol.Biol. 366 (2007) 1447-1458.