A potential antitumor drug (arginine deiminase) reengineered for efficient operation under physiological conditions.
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 mM (M5) and 0.81 mM (M6). The S(0.5) value of M6 (0.81 mM) is lower than that of wild-type PpADI (1.30 mM); 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).
Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
Leilei
L
Zhu
Rajni
R
Verma
Danilo
D
Roccatano
Ye
Y
Ni
Zhi-Hao
ZH
Sun
Ulrich
U
Schwaneberg
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000737:chemistry*; D045744:Cell Line, Tumor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000188:drug therapy; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology
D000970:Antineoplastic Agents; D006867:Hydrolases; C019723:arginine deiminase