Enzimspecifitás megváltoztatása in vitro evolúciós stratégiával = Alteration of enzyme specificity by a strategy based on in vitro evolution

A kutatás célja előző - hasonló című - pályázatunk, amelyben is vitro mesterséges evolúcióval előállítottunk egy megváltozott specifitású DNS-modifikációs metiláz enzimet, kiegészítése és lezárása volt. Elért eredményeink a következőkben foglalhatók össze: 1. Előállítottuk és részlegesen jellemeztük két újabb, szintén megváltozott specifitású mutánsát a korábban vizsgált M.SinI enzimnek. 2. Előállítottuk és részlegesen jellemeztük az M.SinI enzimnek és egyik megváltozott specifitású mutánsának az N-terminális régió első 65 aminosavától megfosztott, rövidített változatát, amelyek működőképesek voltak. 3. Újszerű, az irodalomban egyedülálló, kisérleti rendszert dolgoztunk ki az M.SinI enzim kívánt irányú specifitásváltozásának in vivo detektálására és jellemzésére. | This was the extension and continuation of the work done during our previous OTKA project (with the same title), in which we constructed, by using in vitro directed evolution techniques, a mutant DNA-modification enzyme with changed recognition specificity. The results of the present work could be summarized as follows: 1. By applying the same methods, we constructed two different new mutant variants of the previously studied M.SinI enzyme, with similarly changed recognition specificities, and partially characterized them. 2. We constructed and partially characterized truncated versions of the wild-type and one mutant variant of the M.SinI enzyme, without the N-terminal first 65 amino acids. These truncated enzymes preserved their enzymatic activity. 3. We developed an entirely new experimental system, for the in vivo detection and semiquantitative measurement of the changed recognition specificity of the studied M.SinI enzyme

Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution

The gene coding for the SinI DNA-methyltransferase, a modification enzyme able to recognize and methylate the internal cytosine of the GG(A)/(T)CC sequence, was subjected to in vitro mutagenesis, DNA-shuffling and a strong selection for relaxed GGNCC recognition specificity. As a result of this in vitro evolution experiment, a mutant gene with the required phenotype was selected. The mutant SinI methyltransferase carried five amino acid substitutions. None of these was found in the ‘variable region’ that were thought to be responsible for sequence specificity. Three were located near the N-terminal end, preceding the first conserved structural motif of the enzyme; two were found between conserved motifs VI and VII. A clone engineered to carry out only the latter two replacements (L214S and Y229H) displays relaxed recognition specificity similar to that of the parental mutant, whereas the clone carrying only the N-terminal replacements showed a much weaker change in recognition specificity. The enzyme with two internal mutations was purified and characterized. Its catalytic activity (k(cat)/K(m)) was ∼5-fold lower towards GG(A)/(T)CC and 20-fold higher towards GG(G)/(C)CC than that of the wild-type enzyme