Terminal Uridylyl Transferase: Difference between revisions

Terminal uridylyl transferases (TUTases) belong to a superfamily of polymerase ß nucleotidyl transferases.<ref name="primary citation">PMID:17785418</ref> TUTases have been isolated from ''Trypanosoma brucei'' and also ''Leishmania'' ssp, parasites that cause diseases in humans such as African Sleeping Sickness. It has been suggested that the knowledge of TUTases may aid in the treatment of these diseases as TUTases function in RNA editing in these parasites, and thus can serve as enzymes to target.<ref name="second reference">PMID:11893335</ref> More specifically TUT4 catalyzes a reaction that adds a nucleotide, from a nucleotide triphosphate, to uridine monophosphate (UMP), the minimally required terminal RNA substrate.<ref name="primary citation">PMID:17785418</ref> TUTase4 is able to bind to the nucleotide triphosphates ATP, CTP, GTP or UTP, however, UTP and CTP are preferred, whereas ATP and GTP [[ligand]]s have been shown to cause a significant decrease in enzymatic activity.<ref name="primary citation">PMID:17785418</ref> The preference for UTP causes TUTase4 to typically add a uracil nucleotide to the RNA substrate. This selectivity has a variety of mechanisms, including a loss of coplanarity (π-electron stacking) between the ATP and a tyrosine of the active site (Y189) required for catalysis, and reduced stacking between the UMP and ATP rings.<ref name="primary citation">PMID:17785418</ref> The RNA substrate in trypanosomal TUTases selects for cognate nucleosides and provides a metal ion binding site for Mg²⁺ ions required by the ligand.<ref name="primary citation">PMID:17785418</ref>

TUT4 with a bound <scene name='Reserved_Sandbox_329/Ligand/4'>ATP complex</scene> (consisting of an ATP molecule and two Mg²⁺ ions) has little π-electron stacking with both the active site <scene name='Sandbox_Reserved_329/Tyr189/1'>tyrosine residue</scene> (Y189) and the RNA substrate, and so is destabilizing, however the phosphate groups of the ATP have been shown to superpose well with that of the other ligands.<ref name="primary citation">PMID:17785418</ref> The Mg²⁺ ions are coordinated by three <scene name='Reserved_Sandbox_329/Asp/1'>aspartate residues</scene> (D66, D68, and D136) which are conserved among TUTases, and thus vital in the transferase reaction.<ref name="primary citation">PMID:17785418</ref> Hydrogen bonding and hydrophobic interactions are important in the binding of the RNA substrate to the enzyme as well as the binding of the ligand to the apo protein. Notably, hydrogen bonding interactions occur among <scene name='Sandbox_Reserved_329/Hydrophobic_hbond_interactions/1'>R121, D68, and D136</scene> of TUT4 with the RNA substrate, and among <scene name='Sandbox_Reserved_329/Interactions_atp/1'>S148, Y189, and N147</scene> of the apo protein with the ATP complex.<ref name="primary citation">PMID:17785418</ref> Hydrophobic interactions with the RNA substrate and <scene name='Sandbox_Reserved_329/Hydrophobic_hbond_interactions/1'>V122</scene> of TUT4 also contribute to the transferase reaction.<ref name="primary citation">PMID:17785418</ref> The lack of triple stacking as well as different hydrogen bonding interactions contribute to the preference of TUT4 for UTP instead of ATP, however it is thought that minimal mutations would be required for TUT4 to become ATP specific. <ref name="primary citation">PMID:17785418</ref> The signature active site motif for the polymerase β nucleotidyltransferase superfamily, including TUT4 is hG&nbsp;[G/S]X(9-13)Dh[D/E]h (where X is any amino acid, and h is hydrophobic amino acids).<ref name="second reference">PMID:11893335</ref>