| Abstract [eng] |
Two classes of hydrolase enzymes, the metagenomic cytidine deaminase CDA_F14, disco-vered in the MMB compartment, and the E.coli dCTP deaminase Dcd, have been investigated in the course of this work. By applying a rational design - random and targeted mutagenesis - to both enzymes, the effect of mutations in the corresponding amino acids on both catalytic and substrate activity was analysed. In the CDA_F14 analysis, amino acids N42, E44, Y48, N52 and G54 were changed as they all interact with the ribose ring of the substrate, while the study was interested in substrates with substituents in the ribose ring as some of them may be potential antican-cer/antiviral agents. The results showed that N42 and E44, which affect the 3'-OH group of the ribose, are essential amino acids for the enzyme activity, and that their substitution by alanines significantly reduced the values of the catalytic constants as well as the number of substrates that they can deaminate. Random mutations of Y48 interacting with the 5'-OH group to phenylalanine or tryptophan impaired KM values with 2'-deoxycytidine and N4-benzoyl-2'-deoxycytidine. Howe-ver, the Y48F mutant showed an increased catalytic efficiency constant with N4-benzoyl-2'-deoxycytidine. The amino acids N52 and G54 are thought to form bonds with the 2'-OH group, and mutations in N52I, G54A and the double mutation N52I/G54A also resulted in an increased cataly-tic efficiency constant with the latter substrate. A review of the substrate specificity of all seven mutant proteins showed that substrates with substituents at the 2'-OH position were the most tolerated, whereas substituents with substituents at the 3'-OH position were the least hydrolysed. For one substrate, 5'-levulinyl-N4-benzoyl-2'-deoxycytidine, a change in substrate specificity was observed. The wild-type enzyme did not hydrolyse this compound, whereas the Y48F, Y48W, N52I, G54A and N52I/G54A mutants started to deaminate 5'-levulinil-N4-benzoyl-2'-deoxycytidine. Three mutants, L107A, H121A and H121G, were constructed in the Dcd assay in the light of the information available in the scientific literature, with the expectation that these mutations would have an effect on the activity of Dcd. However, the changes fully inactivated the enzyme, indicating that both amino acids are required for Dcd to function. Since no publications have been published on the activity of wild-type Dcd with modified nucleotides, the present work also tested the activity of Dcd on the latter substrates. The results showed that Dcd did not deami-nate dCTPs with substituents on ribose or heterocyclic base rings. Future plans for a more detailed analysis of Dcd on a wider range of substrates would allow a more reliable confirmation that Dcd is specific only for dCTP. |
