Enzyme Technology Laboratory, Department of Biochemistry, Genetic and Metabolism Research Group, Pasteur Institute of Iran, Tehran, Iran.
Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Appl Microbiol Biotechnol. 2019 Feb;103(4):1725-1735. doi: 10.1007/s00253-018-9529-9. Epub 2019 Jan 3.
Fructosyl peptide oxidase (FPOX, EC 1.5.3) belongs to the family of oxidoreductases, which is used as a diagnostic enzyme for diabetes mellitus. FPOX has activities toward Fru-ValHis and Fru-Lys as model compounds for hemoglobin A1c (HbA1c) and glycated albumin, respectively. However, when the concentration of HbA1c is measured, the activity toward Fru-Lys will cause interference. In this study, we focused on the substrate specificity engineering of FPOX from Eupenicillium terrenum through computational and experimental methods with characteristics more suitable for HbA1c measurement in the blood. Based on structural knowledge of E. terrenum FPOX (PDB ID 4RSL) and molecular modeling results, residues His-377, Arg-62, Lys-380, and Tyr-261 were selected as mutagenesis sites. The best mutant with lower binding energy, stronger hydrophobic interactions, and more hydrogen bonds with Fru-ValHis and higher binding energy toward Fru-Lys was selected for experimental studies. To investigate the conformational changes in FPOX due to the mutation, molecular dynamics simulation was also performed. The genes encoding of native and engineered variants were cloned into pET-22b(+) and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant enzymes were purified and their kinetic properties were studied. Substitution of Tyr261 with Trp resulted in a mutant enzyme with improved specificity for Fru-ValHis, a model compound of HbA1c. The specific activity of mutant FPOX increased by 5.1-fold to 145.2 ± 3.2 U/mg for Fru-ValHis and decreased by 13.7-fold to 1.3 U/mg ± 0.9 for Fru-Lys compared to the native variant. Kinetics analysis indicated that Tyr261Trp FPOX mutant had 11.7-fold increase in K/K for Fru-ValHis compared to the wild-type enzyme, while the K/K for Fru-Lys diminished by 22.4-fold. In summary, our computational and experimental results suggested that the engineered FPOX is a good candidate to efficient determination of HbA1c.
果糖基肽氧化酶（FPOX，EC 1.5.3）属于氧化还原酶家族，被用作糖尿病的诊断酶。FPOX 对 Fru-ValHis 和 Fru-Lys 具有活性，分别作为血红蛋白 A1c（HbA1c）和糖化白蛋白的模型化合物。然而，当测量 HbA1c 的浓度时，Fru-Lys 的活性会引起干扰。在这项研究中，我们通过计算和实验方法，专注于土曲霉来源的 FPOX 的底物特异性工程，以获得更适合血液中 HbA1c 测量的特性。基于土曲霉 FPOX 的结构知识（PDB ID 4RSL）和分子建模结果，选择残基 His-377、Arg-62、Lys-380 和 Tyr-261 作为突变位点。选择具有更低结合能、更强疏水性相互作用和更多氢键与 Fru-ValHis 结合以及更高与 Fru-Lys 结合能的最佳突变体进行实验研究。为了研究由于突变导致的 FPOX 的构象变化，还进行了分子动力学模拟。天然和工程变体的基因被克隆到 pET-22b(+) 中，并在大肠杆菌 BL21（DE3）菌株中生产。表达的重组酶被纯化，并研究了它们的动力学特性。用色氨酸取代 Tyr261 导致突变酶对 Fru-ValHis（HbA1c 的模型化合物）的特异性提高。突变 FPOX 的比活性提高了 5.1 倍，达到 145.2±3.2 U/mg，而对 Fru-Lys 的比活性降低了 13.7 倍，为 1.3 U/mg±0.9。动力学分析表明，与野生型酶相比，Tyr261Trp FPOX 突变体对 Fru-ValHis 的 K/K 增加了 11.7 倍，而对 Fru-Lys 的 K/K 减少了 22.4 倍。总之，我们的计算和实验结果表明，工程化的 FPOX 是高效测定 HbA1c 的良好候选酶。
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