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 N6-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.