| Abstract [eng] |
During this study tetrameric cytidine deaminases exhibiting novel nucleophilic substitution activities in the 4th position of heterocyclic ring i.e., N4-acyl-/N4-alkyl, N4-carboxy, S4-alkyl and O4-alkoxy cytidine substrates converting them to uridine and the according amide, amine, carbamate, thiol, or alcohol were investigated. Before this study these activities of cytidine deaminases were not known. The aim of the study was to, using various molecular modelling techniques, determine structural factors in tetrameric cytidine deaminase CDA_F14, which led to deaminase and deamidase activity against N4 substituted pyrimidine analogues. Initial results revealed that CDA_F14 has a seemingly mobile loop near the active site which could be mainly responsible for the observed activities. This Asp80-Ala86 loop together with the active site and the C-end of the enzyme were investigated using site-directed mutagenesis. The results revealed that the G81L and G85I mutations, when a small aliphatic residue is exchanged to a larger aliphatic residue, lead to a decrease in enzyme activity against both 2’-deoxycytidine and N4-benzoyl-2’-deoxycytidine. The active site cysteine mutations into histidines made the enzyme inactive. The T51G mutants exhibited kinetic parameters that may suggest induced cooperativity between the enzyme’s subunits. The F126A and F126W variants were both active, but the F126W mutant had higher activity because it could still contribute to π-π stacking. Deletions of amino acids in the 127-130 positions don’t lead to major substrate specificity changes, but deletion of the 83-85 residues limits both the enzymes substrate spectrum and overall enzyme efficiency. |
