Discussion - 2qgnA

From cloning of the human tRNA isopentenyltransferase, a C2H2 Zn finger motif is found. From the article by Anna Glovko, this motif is always found in eukaryotic organisms although there are some exceptions for Arabidopsis thaliana, C.elegans and S.pombe. It is surprising to find that this motif is present as a single copy as this motif is usually interacting with more than one zinc finger. Moreover, the common role of this motif is in protein-RNA interation but this might not be the function in eukaryotes. The article suggested that the zinc finger motif may be involved in nuclear retention signal (La Casse 1995) and stability of enzyme conformation (Chong 1995).

Generally, the zinc finger motif have conserved glycine and tryptophan residues along the protein sequence together with cystein and histidine residues at extreme ends involved in coordination with zinc. The best conserved regions found to maintain the structural integrity of the protein in C2H2 zinc fingers are the conserved aliphatic and aromatic residues

Another molecular function of the enzyme is ATP binding. This may be consistent with the fact that a P-loop motif is found to be conserved in humans, E.coli and S. cerevisiea (Anna Glovko 2000). The P-loop is said to be involved in ATP/GTP binding. According to Saraste, this loop together with the zinc finger motif plays a role in ligand binding. Such functional motifs may be from the same superfamily initially but subsequently diverged away from each other. However, there are still some functional regions which are conserved.

From the DALI results, adenylate kinase has structural similarities with tRNA-IPT. The former has a P-loop motif, formed between a β-strand and a α-helix, at its N-terminus. This may mean that tRNA-IPT might have this motif as well. However, the pattern motif of the P-loop in the adenylate kinase is not found in tRNA-IPT. Moreover, guanylate kinase which is also found to have the P-loop does not have the same motif pattern as that of adenylate kinase. Divergence has started then till it reaches tRNA-IPT. Even the structures of both guanylate kinase and tRNA-IPT, when superimposed, have much differences. Thus the P-loop motif in tRNA-IPT is still to be identified but there is a high possibility that the P-motif is located near the ligand binding site.

As found from the structural analysis (PDB and Profunc), the ligand interacts with AVGKT at position 775-780. This is towards the end-terminus of the protein sequence and this sequence is between a β-strand and a α-helix (PDBsum). This supports the fact that the P-loop might be present in tRNA-IPT.

It is surprising that from LOCATE, the mouse enzyme is found in cytosol and mitochondria. According to our knowledge, tRNAs are involved in translation and this process occurs in the cytoplasm. Thus, the enzyme acting on them should be in the cytoplasm as well. However, the human enzyme is found in the nucleus. This may be because the human enzyme has a nuclear signal localization that is also found in yeast IPTs. This is not found in Bacillus halodurans as this signal peptide sequence is found in the additional residues in humans and yeast (Anna Glovko 2000). This may be the reason why human IPT is found in nucleus as well.

There is no literature evidence that tells us the function of the clefts. However, based on the function of the enzyme, there is a possibility that the clefts allow ribosome to bind and interact with this enzyme during translation. According to Laskowski, clefts allow us to know how the proteins are interacting with other molecules on the protein surface. Often, a large and deep cleft is usually associated with the active site of the protein. This means that the red cleft on our protein has the highest possibility of being the active site. However, this is only assumptions and other clefts that are present might be where the active site of the protein as well.

The DALI results obtained showed that there are several other enzymes that are structurally related to tRNA-IPT. They are isopentenyl transferase, guanylate kinase and adenylate kinase, starting from the most similar structure. According to biochemist view, similar structure proteins may contribute to similar functions as well. Thus, the functions of these enzymes are studied to further understand and support the existing function of our protein.

Using InterPro, the information on the functions of these enzymes is retrieved. Guanylate kinase catalyzes the ATP-dependent phosphorylation of GMP into GDP while adenylate kinase catalyse the Mg-dependent reversible conversion of ATP and AMP to two molecules of ADP, an essential reaction for many processes in living cells. Both of these enzymes may act on different molecules but the reactions that they catalyse involve phosphorylation.

The ability to phosphorylate is lost in isopentenyl transferases, which is also known as dimethylallyl transferase. This enzyme adds the isopentenyl group on adenine base situated at the 5'-terminal phosphate group. So far, tRNA-IPT only has the function of isopentenyl transferase and does not exhibit any phosphorylation function. However, there are still possibilities that tRNA-IPT has the ability to phosphorylate.

Expression of tRNA-IPT is higher in several tissues but generally it is expressed in all cells. This is to ensure that efficient and correct translation takes place in each cell. This enzyme is particularly high in oocyte and adipose tissue. The former is constantly undergoing cell division and differentiation while the latter is often involved in energy production, energy storage, and hormone production. All of these processes require high levels of enzyme activity and the presence of different adaptor molecules and transcription factors.