Aspergillus nidulans thermostable arginine deiminase-Dextran conjugates with enhanced molecular stability, proteolytic resistance, pharmacokinetic properties and anticancer activity

• Published in: Enzyme and microbial technology 2019-12, Vol.131, p.109432, Article 109432
• Main Authors: El-Sayed, Ashraf S.A., Shindia, Ahmed A., Zeid, Azza A. Abou, Yassin, Amany M., Sitohy, Mahmoud Z., Sitohy, Basel
• Format: Article
• Language: English
• Subjects: Animals; Anticancer activity; Antineoplastic Agents - chemistry; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacokinetics; Antineoplastic Agents - pharmacology; Arginine - metabolism; Aspergillus nidulans; Aspergillus nidulans - enzymology; Cell Line, Tumor; Cell Proliferation; Citrulline - metabolism; Dextran conjugation; Dextrans - metabolism; Enzyme Stability; Humans; Hydrogen-Ion Concentration; Hydrolases - chemistry; Hydrolases - metabolism; Hydrolases - pharmacokinetics; Hydrolases - pharmacology; Kinetics; Mice; Molecular Weight; Pharmacokinetics; Protein Multimerization; Proteolysis; Temperature; Thermostable arginine deiminase
• ISSN: 0141-0229; 1879-0909; 1879-0909
• DOI: 10.1016/j.enzmictec.2019.109432

•Aspergillus nidulans ADI is a homotrimer of molecular subunit structure 48 kDa.•Dex-ADI conjugate has more resistance to proteinase K proteolysis by 2.5 folds.•The catalytic citrullinating affinity of Dex-ADI to free L-arginine was increased by 5.3 folds.•The activity of Dex-ADI against breast, liver and colon cancers had been increased by 1.7 folds.•The titer of L-arginine was reduced by 2.5 folds with Dex-ADI in vivo. The potential anticancer activity of arginine deiminase (ADI) via deimination of l-arginine into citrulline has been extensively verified against various arginine-auxotrophic tumors, however, the higher antigenicity, structural instability and in vivo proteolysis are the major challenges that limit this enzyme from further clinical implementation. Since, this clinically applied enzyme was derived from Mycobacterium spp, thus, searching for ADI from eukaryotic microbes “especially thermophilic fungi” could have a novel biochemical, conformational and catalytic properties. Aspergillus nidulans ADI was purified with 5.3 folds, with molecular subunit structure 48 kDa and entire molecular mass 120 kDa, ensuring its homotrimeric identity. The peptide fingerprinting analysis revealing the domain Glu95-Gly96-Gly97 as the conserved active site of A. nidulans ADI, with higher proximity to Mycobacterium ADI clade IV. In an endeavor to fortify the structural stability and anticancer activity of A. nidulans ADI, the enzyme was chemically modified with dextran. The optimal activity of Dextran-ADI conjugates was determined at 0.08:20 M ratio of ADI: Dextran, with an overall increase to ADI molecular subunit mass to ˜100 kDa. ADI was conjugated with dextran via the ε-amino groups interaction of surface lysine residues of ADI. The resistance of Dextran-ADI conjugate to proteolysis had been increased by 2.5 folds to proteinase K and trypsin, suggesting the shielding of >50% of ADI surface proteolytic recognition sites. The native and Dextran-ADI conjugates have the same optimum reaction temperature (37 °C), reaction pH and pH stability (7.0–8.0) with dependency on K+ ions as a cofactor. Dextran-ADI conjugates exhibited a higher thermal stability by ˜ 2 folds for all the tested temperatures, ensuring the acquired structural and catalytic stability upon dextran conjugation. Dextran conjugation slightly protect the reactive amino and thiols groups of surface amino acids of ADI from amino acids suicide inhibitors. The affinity of ADI was increased by 5.3 folds