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

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 effecters 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 subsuates 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. (C) 2000 Elsevier Science B.V. All rights reserved.