This page was created for an undergraduate course at Davidson College.
Orthologs of S. cerevisiae isocitrate dehydrogenase
Yeast have five different isocitrate dehydrogenase genes (IDH1, IDH2, IDP1, IDP2, IDP3). IDH1 and IDH2 form a heterodimer located in the mitochondrial matrix (Swiss-Prot, P28834, P28241). The IDH1-IDH2 complex is involved in glutamate biosynthesis, isocitrate metabolism, tricarboxylic acid cycle (SGD, IDH1, IDH2). The complex catalyzes the following reaction (Swiss-Prot, P28834, P28241):
Isocitrate + NAD(+) = 2 oxoglutarate + CO(2) + NADH.
The heterodimer is regulated by AMP, NAD+, and citrate (Swiss-Prot, P28834, P28241).
IDP1 and IDP2 are both involved in glutamate biosynthesis and isocitrate metabolism (SGD, IDP1, IDP2). Each enzyme acts as a homodimer and catalyzes the following reaction (Swiss-Prot,P21954, P41939):
isocitrate + NADP(+)= 2 oxoglutarate + CO(2) + NADPH.
IDP1 is located in the mitochondria and regulates the TCA cycle and respiration (Swiss-Prot, P21954). IDP2 is located in the cytoplasm (Swiss-Prot, P41939).
IDP3 is involved in NADP regeneration, fatty acid beta oxidation, and isocitrate metabolism (SGD, IDP3). It catalyzes the following reaction (Swiss-Prot, P53982):
isocitrate + NADP(+) = 2 oxoglutarate + CO(2) + NADPH.
IDP3 is located in the peroxisomes and produces NADPH for fatty acid and sterol synthesis (Swiss-Prot, P53982).
Pictures of similar enzymes whose structure has been isolated:
Taken from PDB.
No isocitrate dehydrogenase proteins that complex with NAD have been isolated. Isopropylmalate dehydrogenase is in the same family of enzymes as isocitrate dehydrogenase (Swiss-Prot, P28834, P28241). An isopropylmalate dehydrogenase that bind to NAD has been isolated.
isocitrate dehydrogenase complexed with NADP+, isocitrate, and calcium (1ai2)
In lab, my molecular biology class is trying to clone all 5 isocitrate dehydrogenase genes and then express the proteins using the pQE-30UA expression vector kit. My lab partner and I were assigned the IDP3 gene. For this assignment we were told to find 5 orthologs of our gene. To do this I ran my protein sequence through the BLAST program.
What is the BLAST program?
For a more detailed explanation go to the following link BLAST overview.
BLAST is a program that tries to find similar sequences by comparing two sequences at a time.
How does BLAST work?
For a more detailed explanation go to the following link BLAST course.
First you enter a query sequence. Then the program compares your sequence with all the sequences in the database two at a time. The score represents how closely the sequences aligned. The higher the score the better the alignment. The E-value is the number of hits one would expect with scores greater than or equal to the score retrieved by chance alone. The lower the E-vaule the more similar the two sequences are likely to be. Thus you want high scores and low E-values. If you compare two identical sequences the E-value will be 0.
Results of my BLAST search
When the IDP3 amino acid sequence was run through the BLASTP program at NCBI many orthologs were retrieved. (Full BLASTp result report.) Isocitrate dehydrogenase is a basic enzyme used by organisms across all taxa. Many of the hits were found in model organisms. Since research using humans is limited, finding similar sequences in model organisms allows scientists to learn more about enzymes in humans. Below are six orthologs from taxa ranging from bacteria, a single cell prokaryote, to humans, a multi-cell eukaryote The scores and E-values for each match with IDP3 from yeast are also given.
(Click on the link to see the cDNA sequence and amino acid sequence for each ortholog.)
Score: 571 bits
- E-value: 6e-162
Bacteria-a-protepobacteria-prokaryote
Score: 509 bits
E-value: 1e-141
Score: 513 bits
- E-value: 1e-144
Rice-plant-monocot
Score: 533 bits
E-value: 1e-150
Peach-plant-eudicots
Score: 525 bits
- E-Value: 4E-148
Mouse-mammal
Score: 527 bits
- E-value: 7e-149
Human-mammal
Score: 525 bits
E value: 3e-148
The cloning of the 5 isocitrate genes did not go as planned. Many of the plasmids did not have inserts or if they did have inserts, all the inserts were in the reverse orientation. Thus we only had a limited number of bacteria with expression vectors that had inserts in the correct orientation and thus would express a functional protein. We don't know for sure why some of the inserts did not clone into the expression vectors. The expression vectors were not supposed to self ligate on each other but apparently some did. We knew that some of the vectors would have inserts in the reverse orientation because both cloning ends of the insert were identical. To ensure that inserts only clone into a plasmid in the forward orientation each of the ends of the insert must have a different restriction site.
contact: lirobinson@davidson.edu