Hydrolytic cleavage of N-6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases

0324946 - ÚEB 2009 RIV GB eng J - Článek v odborném periodiku
Pospíšilová, H. - Šebela, M. - Novák, Ondřej - Frébort, I.
Hydrolytic cleavage of N-6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases.
[Hydrolytické štěpení N6-substituovaných derivátů adeninu eukaryontními adenin- a adenosindeaminasami.]
Bioscience Reports. Roč. 28, č. 6 (2008), s. 335-347. ISSN 0144-8463. E-ISSN 1573-4935
Grant CEP: GA ČR(CZ) GA522/06/0022
Výzkumný záměr: CEZ:AV0Z50380511
Klíčová slova: adenine deaminase * adenosine deaminase (ADA) * aminohydrolase
Kód oboru RIV: EB - Genetika a molekulární biologie
Impakt faktor: 2.525, rok: 2008

Synopsis Homogeneous adenine deaminases (EC 3.5.4.2) from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and a putative ADA (adenosine deaminase; EC 3.5.4.4) from Arabidopsis thaliana were obtained for the first time as purified recombinant proteins by molecular cloning of the corresponding genes and their overexpression in Escherichia coli. The enzymes showed comparable molecular properties with well-known mammalian ADAs, but exhibited much lower k(cat) values. Adenine was the most favoured substrate for the yeast enzymes, whereas the plant enzyme showed only very low activities with either adenine, adenosine, AMP or ATP Interestingly, the yeast enzymes also hydrolysed NB-Substituted adenines from cytokinins, a group of plant hormones, cleaving them to inosine and the corresponding side chain amine. The hydrolytic cleavage of synthetic cytokinin 2,6-di-substituted analogues that are used in cancer therapy, such as olomoucine, roscovitine and bohemine, was subsequently shown for a reference sample of human ADA1. ADA1, however, showed a different reaction mechanism to that of the yeast enzymes, hydrolysing the compounds to an adenine derivative and a side chain alcohol. The reaction products were identified using reference compounds on HPLC coupled to UV and Q-TOF (quadrupole-time-of-flight) detectors. The ADA1 activity may constitute the debenzylation metabolic route already described for bohemine and, as a consequence, it may compromise the physiological or therapeutic effects of exogenously applied cytokinin derivatives.

Expresí v E. coli byly připraveny tři rekombinantní enzymy - adenindeaminasy (EC 3.5.4.2) z kvasinek Saccharomyces cerevisiae a Schizosaccharomyces pombe a domnělá adenosindeaminasa ( EC 3.5.4.4) z Arabidopsis thaliana. Tyto enzymy byly charakterizovány enzymologickými postupy a bylo zjištěno, že mají podobné molekulové vlastnosti jako savčí adenosindeaminasy, ale přitom podstatně nižší katalytickou aktivitu. Enzymy z kvasinek působily kromě adeninu i na N6-substituované deriváty adeninu ze skupiny cytokininů. S použitím referenční lidské adenosindeaminasy byl studován hydrolytický účinek vůči 2,6-disubstituovaným cytokininovým analogům, které se používají v protinádorové terapii. Byl zjištěn odlišný mechanismus hydrolýzy.
Trvalý link: http://hdl.handle.net/11104/0172521