A novel thymidine phosphorylase to synthesize (halogenated) anticancer and antiviral nucleoside drugs in continuous flow.

Four pharmaceutically relevant nucleoside analogues (5-fluoro-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-iodo-2'-deoxyuridine) have been synthesized by using a novel thymidine phosphorylase from the halotolerant H. elongata (HeTP). Following enzyme immobilization on microbeads, the biocatalyst was implemented as a packed-bed reactor for the continuous production of halogenated nucleosides, achieving up to 90% conversion at the 10 mM scale with 30 min residence time. Taking the synthesis of floxuridine (5-fluoro-2'-deoxyuridine) as a study case, we obtained the highest space-time yield (5.5 g L-1 h-1) reported to date. In addition, bioinformatic tools such as MD analysis and CapiPy have contributed to shine light on the catalytic performance of HeTP as well as its immobilization, respectively.

Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases.

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.