Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase as biocatalyst in the preparative synthesis of 2′-deoxyribonucleoside-5′-triphosphates

Abstract

Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase has been purified and evaluated as a biocatalyst for the preparative synthesis of 2′-deoxyribonucleoside-5′-triphosphates. The addition of expensive 2′-deoxyribonucleoside-5′-triphosphates as allosteric effectors of ribonucleosidetriphosphate reductase was not necessary due to high concentrations of inorganic salts in the reaction mixture. Good conversion of the tested ribonucleoside-5′-triphosphate substrates ATP, CTP, GTP, ITP, and UTP was observed. From a variety of reducing agents 1,4-dithio- dl-threitol (DTT), 1,4-dithioerythritol (DTE), bis-(2-mercaptoethyl)-sulfone, and 1,3-propanedithiol showed to be the most effective reducing agents for re-reduction of the active center thiols of ribonucleosidetriphosphate reductase. The kinetic parameters of ribonucleosidetriphosphate reductase with respect to affinity of ribonucleoside-5′-triphosphate substrates, the cofactor 5′-deoxyadenosylcobalamin, and the reducing agents DTT or 1,3-propanedithiol under the employed reaction conditions were determined. Substrate inhibition was not observed. Preparative gram-scale 2′-reductions of ribonucleoside-5′-triphosphates proceeded to completion.