Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides

Fruit fly deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and r.t. for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraA-MP). Upon optimization of the reaction conditions (50 mM ammonium acetate, ratio substrate/ATP 1:1.25, 2 mM MgCl2, 37 °C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved. Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).