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Keywords:

  • arginine deiminase;
  • directed evolution;
  • protein engineering;
  • thermal resistance

Abstract

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine-auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas. Studies in human lymphatic leukemia cell lines have confirmed the anti-angiogenic activity of ADI. Activity and thermal resistance limit the efficacy of ADI in treatment of auxotrophic tumors. Previously, we reengineered ADI from Pseudomonas plecoglossicida (PpADI) for improved activity under physiological conditions (37 °C, PBS buffer, pH 7.4) by two rounds of directed evolution and combination of beneficial substitutions through site-directed mutagenesis. The best variant, PpADI M6 (K5T/D38H/D44E/A128T/E296K/H404R), showed a 64.7-fold improvement in kcat value and a 37.6 % decreased S0.5 value under physiological conditions. However, M6 lost rapidly its activity (half-life of ∼2 days at 37 °C). Here we report the re-engineering of PpADI M6 for improved thermal resistance by directed evolution in order to increase its half-life under physiological conditions. Directed evolution and recombination of the two most beneficial positions yielded variant PpADI M9 (K5T/D38H/D44E/A128T/V140L/E296K/F325L/H404R), for which the Tm value increased from 47 (M6) to 54 °C (M9); this corresponds to an increased half-life from ∼2 days (M6) to ∼3.5 days (M9) under physiological conditions. Structure analysis of the homology model of M9 showed that the beneficial substitutions V140L and F325L likely promote the formation of tetrameric PpADI, which has greater thermal resistance than dimeric PpADI.