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Updated: May 4, 2026
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Protein engineering of the antitumor enzyme PpADI for improved thermal resistance
Leilei Zhu1, Feng Cheng, Victoria Piatkowski
1Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52056 Aachen (Germany).
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 k(cat) value and a 37.6% decreased S(0.5) 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 T(m) 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.
Insights
Arginine deiminase (ADI) was engineered for improved thermal stability, enhancing its potential as an antitumor drug. The new variant, PpADI M9, shows increased half-life and thermal resistance for treating arginine-auxotrophic tumors.
Area of Science:
- Biochemistry and Molecular Biology
- Enzyme Engineering
- Cancer Therapeutics
Background:
- Arginine deiminase (ADI) is a promising antitumor agent for arginine-auxotrophic cancers like HCC and melanoma.
- Current ADI efficacy is limited by its activity and thermal stability under physiological conditions.
- Previous engineering efforts improved PpADI activity but resulted in a variant (M6) with a short half-life.
Purpose of the Study:
- To re-engineer PpADI M6 for enhanced thermal resistance and increased half-life under physiological conditions.
- To improve the therapeutic potential of ADI for treating arginine-dependent tumors.
Main Methods:
- Directed evolution was employed to improve the thermal stability of the previously engineered PpADI M6 variant.
- Site-directed mutagenesis and recombination were used to combine beneficial mutations.
- Homology modeling was utilized to analyze structural changes in the engineered enzyme.
Main Results:
- A new variant, PpADI M9, exhibited a significant increase in melting temperature (Tm) from 47°C to 54°C.
- PpADI M9 demonstrated an extended half-life from approximately 2 days to 3.5 days at 37°C.
- Structural analysis suggests that mutations V140L and F325L promote a more stable tetrameric form of ADI.
Conclusions:
- The engineered PpADI M9 variant possesses improved thermal resistance and stability.
- This enhanced stability increases the potential therapeutic efficacy of ADI for cancer treatment.
- The tetrameric structure of M9 likely contributes to its superior thermal stability compared to dimeric forms.
