Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
Appl Microbiol Biotechnol. 2013 Oct;97(19):8599-607. doi: 10.1007/s00253-013-4705-4. Epub 2013 Jan 26.
Amadori compounds and their cross-linked products have been implicated in diabetic complications and some age-related diseases. Fructosyl amine oxidases (FAOXs) are a family of enzymes that can cleave the amadori compounds. However, the natural enzymes are only active on small substrates (fructosyl amino acids or dipeptides), which limits the therapeutic and diagnostic applications of these enzymes. In this study, amadoriase II, a member of the FAOX family from Aspergillus fumigatus was engineered to broaden its substrate range using a modified combinatorial active site saturation testing approach. The two loops at the entrance of the substrate channel were targeted. Saturation mutagenesis was carried out to search for hot-spot sites, followed by pairwise mutagenesis and subsequent combination of active mutations. Five sites on the loops were found to be critical for accessibility for two model bulky substrates, fructosyl adamantanamine and fructosyl-polylysine (3-13 lysines). Two best mutants (with three and five mutations, respectively) were obtained, with a specific activity toward the model substrates 20.6-fold and 16.8-fold that of the wild-type, respectively. Deconvolution experiments revealed the cooperativity of the mutations.
阿玛多里化合物及其交联产物与糖尿病并发症和一些与年龄相关的疾病有关。果糖胺氧化酶（FAOXs）是能够切割阿玛多里化合物的酶家族。然而，天然酶仅对小底物（果糖基氨基酸或二肽）具有活性，这限制了这些酶的治疗和诊断应用。在这项研究中，使用改良的组合活性位点饱和测试方法，对来自烟曲霉的 FAOX 家族成员 Amadoriase II 进行了工程改造，以拓宽其底物范围。靶向底物通道入口处的两个环。进行饱和诱变以寻找热点，然后进行成对诱变，随后组合活性突变。在这两个环上发现了五个对两个模型大体积底物（果糖基金刚烷胺和果糖基聚赖氨酸（3-13 个赖氨酸））的可及性至关重要的位点。发现了两个最佳突变体（分别具有三个和五个突变），它们对模型底物的比活性分别是野生型的 20.6 倍和 16.8 倍。去卷积实验揭示了突变的协同作用。
