Hydrophobic Mutagenesis and Semi-rational Engineering of Arginine Deiminase for Markedly Enhanced Stability and Catalytic Efficiency.

Due to its systemic arginine degradation, arginine deiminase (ADI) has attracted attentions as an anti-tumor drug. Its low activity at physiological conditions among other limitations has necessitated its engineering for improved properties. The present study describes the hydrophobic mutagenesis and semi-rational engineering of ADI from Pseudomonas plecoglossicida (PpADI). Using an improved ADI variant M13 (D38H/A128T/E296K/H404R/I410L) as parent, site saturation mutagenesis at position 162 resulted in an over 20 % increase in protein solubility. Compared with M13 (15.23 U/mg), mutants M13-2 (M13+S245D) and M13-5 (M13+R243L) exhibited enhanced specific activity of 21.19 and 31.20 U/mg at physiological conditions. M13-5 displayed enhanced substrate specificity with a dramatic reduction in its K m value (from 0.52 to 0.16 mM). It is speculated that the improvements in M13-5 could mainly be attributed to the enhanced structural stability due to an R243L substitution. The hydrophobic contribution of Leu 243 was supported by mutant M13-9 (M13+A276W) generated based on the hydrophobic mutagenesis concept. M13-9 showed a specific activity of 18.68 U/mg, as well as remarkable thermal and pH stability. It retained over 90 % activity over pH range from 4.5 to 8.5. At 60 °C, the half-life of M13-9 was enhanced from 4 to 17.5 min in comparison with M13, and its specific activity at 62 °C (93.0 U/mg) was approximately fivefold of that determined at 37 °C. Our results suggest that the increased hydrophobicity around the active regions of PpADI might be crucial in improving its structural stability and ultimately catalytic efficiency.

PEGylation and pharmacological characterization of a potential anti-tumor drug, an engineered arginine deiminase originated from Pseudomonas plecoglossicida.

Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for arginine-auxotrophic tumors. PEGylation is one of the best methods to formulate a bioconjugated protein with extended physical stability and reduced immunogenicity. Here, PEGylation and pharmacological properties of an engineered ADI originated from Pseudomonas plecoglossicida were studied. Among polyethylene glycol (PEG) reagents with succinimidyl ester groups varying in size and linkers, three PEGylated products with high yield and catalytic activity were further characterized, named ADI-SS(20 kDa), ADI-SC(20 kDa), and ADI-SPA(20 kDa). In the pharmacodynamic/pharmacokinetic (PD/PK) studies with ADI-SPA(20 kDa), a remarkable improvement in circulating half-life compared with native ADI was observed. ADI-SPA(20 kDa) injections via intravenous, intramuscular and subcutaneous routes all exhibited superior efficacy than native ADI on depleting serum arginine. Additionally, our results demonstrated that single ADI-SPA(20 kDa) administration of 5 U/mouse via intravenous injection could maintain serum arginine at an undetectable level for 5 days with a half-life of 53.2 h, representing 11-fold improvement in half-life than that of the native ADI. In a mice H22 hepatocarcinoma model, ADI-SPA(20 kDa) dosage of 5 U per 5 days showed an inhibition rate of 95.02% on tumor growth during 15-day treatments.