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 to free L-arginine with a dramatic reduction in citrullination of peptidylarginine residues upon dextran conjugation. The anticancer activity of ADI to breast (MCF-7), liver (HepG-2) and colon (HCT8, HT29, DLD1 and LS174 T) cancer cell lines was increased by 1.7 folds with dextran conjugation in vitro. Pharmacokinetically, the half-life time of ADI was increased by 1.7 folds upon dextran conjugation, in vivo. From the biochemical and hematological parameters, ADIs had no signs of toxicity to the experimental animals. In addition to the dramatic reduction of L-arginine in serum, citrulline level was increased by 2.5 folds upon dextran conjugation of ADI. This is first report exploring thermostable ADI from thermophilic A. nidulans with robust structural stability, catalytic efficiency and proteolytic resistance.

中文翻译：

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构巢曲霉耐热精氨酸脱亚胺酶-葡聚糖偶联物，具有增强的分子稳定性、蛋白水解抗性、药代动力学特性和抗癌活性

精氨酸脱亚胺酶 (ADI) 通过将 L-精氨酸脱亚为瓜氨酸的潜在抗癌活性已被广泛证实可对抗各种精氨酸营养缺陷型肿瘤，然而，较高的抗原性、结构不稳定性和体内蛋白水解是限制这种酶从进一步临床实施。由于这种临床应用的酶来源于分枝杆菌属，因此，从真核微生物“特别是嗜热真菌”中寻找 ADI 可能具有新的生化、构象和催化特性。构巢曲霉 ADI 纯化 5.3 倍，分子亚基结构为 48 kDa，整个分子质量为 120 kDa，确保其同源三聚体身份。肽指纹分析揭示结构域 Glu95-Gly96-Gly97 是构巢曲霉 ADI 的保守活性位点，与分枝杆菌 ADI 进化枝 IV 的接近度更高。为了加强构巢曲霉 ADI 的结构稳定性和抗癌活性，该酶用葡聚糖进行了化学修饰。葡聚糖-ADI缀合物的最佳活性在ADI:葡聚糖的0.08:20M比率下确定，ADI分子亚基质量总体增加至～100kDa。ADI 通过 ADI 表面赖氨酸残基的 ε-氨基相互作用与葡聚糖缀合。葡聚糖-ADI 偶联物对蛋白酶 K 和胰蛋白酶的抗性增加了 2.5 倍，表明屏蔽了 >50% 的 ADI 表面蛋白水解识别位点。天然和葡聚糖-ADI 偶联物具有相同的最佳反应温度 (37 °C)、反应 pH 和 pH 稳定性 (7.0-8.0)，依赖于 K+ 离子作为辅助因子。葡聚糖-ADI 偶联物在所有测试温度下都表现出约 2 倍的更高热稳定性，确保在葡聚糖偶联后获得结构和催化稳定性。葡聚糖缀合略微保护 ADI 表面氨基酸的反应性氨基和硫醇基团免受氨基酸自杀抑制剂的影响。ADI 对游离 L-精氨酸的亲和力增加了 5.3 倍，葡聚糖缀合后肽基精氨酸残基的瓜氨酸化显着减少。ADI 对乳腺癌 (MCF-7)、肝脏 (HepG-2) 和结肠 (HCT8、HT29、DLD1 和 LS174 T) 癌细胞系的抗癌活性在体外与葡聚糖结合时增加了 1.7 倍。在药代动力学上，在体内，葡聚糖结合后 ADI 的半衰期增加了 1.7 倍。从生化和血液学参数来看，ADIs对实验动物没有毒性迹象。除了血清中 L-精氨酸的显着减少外，ADI 与葡聚糖结合后瓜氨酸水平增加了 2.5 倍。这是第一份探索来自嗜热 A. nidulans 的热稳定 ADI 的报告，该 ADI 具有强大的结构稳定性、催化效率和蛋白水解抗性。ADI 的葡聚糖缀合后 5 倍。这是第一份探索来自嗜热 A. nidulans 的热稳定 ADI 的报告，该 ADI 具有强大的结构稳定性、催化效率和蛋白水解抗性。ADI 的葡聚糖缀合后 5 倍。这是第一份探索来自嗜热 A. nidulans 的热稳定 ADI 的报告，该 ADI 具有强大的结构稳定性、催化效率和蛋白水解抗性。