title: Rapid evolution of arginine deiminase for improved anti-tumor activity.
abstract: Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K(m) value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100-120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime-thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K(m) under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K(m) value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Rd., 214122, Wuxi, People's Republic of China. yni@jiangnan.edu.cn
authors.forename: Ye
authors.initials: Y
authors.lastname: Ni
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Liu
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
authors.forename: Leilei
authors.initials: L
authors.lastname: Zhu
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Lifeng
authors.initials: L
authors.lastname: Li
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase