Chime Images of IDH and Orthologs

"This web page was produced as an assignment for an undergraduate course at Davidson College."


Figure 1.  Chime figure of Isocitrate Dehydrogenasae complexed with isocitrate, NADP+, and calcium.  This is from E. coli. http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=6723
 

 

Figure 2.  Isocitrate Dehydrogenase Complex With NADP+.  This chime is also from E. coli.
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=3431

NADP+, which is a component of the above figure, is the coenzyme for IDP1, IDP2, and IDP3.
 

Because no NAD+ chime images were found when searching under "isocitrate dehydrogenase", it was necessary to expand the search to include such things as glycerol dehydrogenase.
 

Figure 3.  Bacillus Stearothermophilus Glycerol Dehydrogenase Complex With Nad+.
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=17260

NAD+ is a component of the above figure and is the coenzyme for IDH1 and IDH2.

The above chime figures illustrate NADP and NAD complexed with other molecules. It was difficult to find a variety of organisms for the chime figures and so there was no way to combine the information given by the chimes with that from the orthologs. 


IDP3
After doing a BLAST search in Genbank for 5 orthologs to IDP3, few organisms were found that had significant sequence similarities.   Most of the organisms were nothing other than S. cerevisiae.  There were no orthologs that matched the organisms found for the chime images. 

Organism: Kluyveromyces lactis which is a eukaryotic fungi
Amino Acid and Protein Sequence for the CDS:
http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/getfeat?gi=12802048&id=1&entity=465

Organism: Candida tropicalis which is also an eukaryotic fungi
Amino Acid and Protein Sequence for the CDS:
http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/getfeat?gi=2541873&id=1&entity=2

Organism: Solanum tuberosum which is a potato.
Amino Acid and Protein Sequence for the CDS:
http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/getfeat?gi=517487&id=1&entity=31

Organism: Mus musculus or house mouse
Amino Acid and Protein Sequence for the CDS:
http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/getfeat?gi=6754277&id=1&entity=2

Organism: Homo sapiens
Amino Acid and Protein Sequence for the CDS: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/getfeat?gi=18552191&id=1&entity=1
 


Summary...

NAD-specific isocitrate dehydrogenase is an octamer composed of the subunits IDH1 and IDH2.  These two subunits are not identical but are similar (Panisko and McAlister 2001).  In Saccharomyces cerevisiae, the metabolic degradation of saturated fatty acids is exclusively confined to peroxisomes.  The IDP3 gene product is a yeast peroxisome NADP-dependent isocitrate dehydrogenase.  Yeast cells lacking IDP3 will grow on saturated fatty acids, but growth is impaired on unsaturated fatty acids.  This indicates that IDP3 is involved in their metabolic utilization (Henke et al. 1998).  Many of the species in which there were orthologs did not show that this sequence related to the citric acid cycle. But these entries in the BLAST search did not match with the inserted sequence to a high degree. The orthologs listed above from varying species (no prokaryote was found) are all isocitrate dehydrogenase. Thus, it does appear that this is a conserved domain.

In the Lab...

In lab, my lab partner and I have not been able to conclusively determine that we have cloned our targeted gene-- IDP3. We digested our insert first with the restricion enzymes Bam and HindIII in order to comfirm that we have an insert. The reason we did this intial step was because other lab groups also had difficulty proving they had an IDP3 insert in the forward direction. Thus we acted conservatively. Then, once determining that we did have an insert, we digested with the enzyme pairs Stu and Dra, Sal and NcoI, and Sty hoping that these combinations would in fact tell us our insert and possibly orientation. But unfortunately, our digestions showed that our insert is not IDP3. We are now doing a Southern Blot in order to determine what our insert is in fact.


References

Henke B., Girzalsky W., Berteaux-Lecellier V., and Erdmann R. 1998. IDP3 encodes a peroxisomal NADP-dependent isocitrate dehydrogenase required for the beta-oxidation of unsaturated fatty acids. J Biol Chem. 273(6):3702-11. <http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9452501&dopt=Abstract>

Panisko E.A., McAlister-Henn L. 2001. Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase. J Biol Chem.
276(2):1204-10. <http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11042198&dopt=Abstract>

Home

Molecular Front Page

Davidson College

For questions or comments contact elsellars@davidson.edu