Premium
A Potential Antitumor Drug (Arginine Deiminase) Reengineered for Efficient Operation under Physiological Conditions
Author(s) -
Zhu Leilei,
Verma Rajni,
Roccatano Danilo,
Ni Ye,
Sun ZhiHao,
Schwaneberg Ulrich
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000458
Subject(s) - arginine deiminase , drug , arginine , chemistry , pharmacology , biochemistry , medicine , amino acid
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, and studies on human lymphatic leukemia cell lines have confirmed that ADI has antiangiogenic activity. Recent studies showed that a combination of taxane and ADI‐PEG20, which induces caspase‐independent apoptosis, is more effective than taxane monotherapy for prostate cancer. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) and of many other ADI enzymes lies in their pH‐dependent activity profile. PpADI has a pH optimum at 6.5 and a pH shift from 6.5 to 7.5 results in an ∼80 % activity drop (the pH of human plasma is 7.35 to 7.45). In 2010, we reported a proof of concept for ADI engineering by directed evolution that resulted in variant M2 (K5T/D44E/H404R). M2 has a pH optimum of pH 7.0, a fourfold higher k cat value than the wild‐type PpADI (pH 7.4, 0.5 M phosphate buffer), and an increased K m value for substrate arginine. In our latest work, variants M5 (K5T/D38H/D44E/A128T/H404R) and M6 (K5T/D38H/D44E/A128T/E296K/H404R) were generated by directed evolution by employing PBS buffer (pH 7.4), which mimics physiological conditions. The S 0.5 value of parent M3 (K5T/D44E/A128T/H404R) decreased from 2.01 to 1.48 m M (M5) and 0.81 m M (M6). The S 0.5 value of M6 (0.81 m M ) is lower than that of wild‐type PpADI (1.30 m M ); the k cat values improved from 0.18 s −1 (wild‐type PpADI) to 17.56 s −1 (M5, 97.6‐fold) and 11.64 s −1 (M6, 64.7‐fold).