Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan
Asano Active Enzyme Molecule Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Imizu, Toyama, Japan.
Appl Environ Microbiol. 2019 May 30;85(12). doi: 10.1128/AEM.00459-19. Print 2019 Jun 15.
Following the evolutionary track of enzymes can help elucidate how enzymes attain their characteristic functions, such as thermal adaptation and substrate selectivity, during the evolutionary process. Ancestral sequence reconstruction (ASR) is effective for following evolutionary processes if sufficient sequence data are available. Selecting sequences from the data to generate a curated sequence library is necessary for the successful design of artificial proteins by ASR. In this study, we tried to follow the evolutionary track of l-arginine oxidase (AROD), a flavin adenine dinucleotide (FAD)-dependent amino acid oxidase (LAAO) that exhibits high specificity for l-arginine. The library was generated by selecting sequences in which the 15th, 50th, 332nd, and 580th residues are Gly, Ser, Trp, and Thr, respectively. We excluded sequences that are either extremely short or long and those with a low degree of sequence identity. Three ancestral ARODs (AncARODn0, AncARODn1, and AncARODn2) were designed using the library. Subsequently, we expressed the ancestral ARODs as well as native AROD (OkAROD) in bacteria. AncARODn0 is phylogenetically most remote from OkAROD, whereas AncARODn2 is most similar to OkAROD. Thermal stability was gradually increased by extending AROD sequences back to the progenitor, while the temperature at which the residual activity is half of the maximum measured activity () of AncARODn0 was >20°C higher than that of OkAROD. Remarkably, only AncARODn0 exhibited broad substrate selectivity similar to that of conventional promiscuous LAAO. Taken together, our findings led us to infer that AROD may have evolved from a highly thermostable and promiscuous LAAO. In this study, we attempted to infer the molecular evolution of a recently isolated FAD-dependent l-arginine oxidase (AROD) that oxidizes l-arginine to 2-ketoarginine. Utilizing 10 candidate AROD sequences, we obtained a total of three ancestral ARODs. In addition, one native AROD was obtained by cloning one of the candidate ARODs. The candidate sequences were selected utilizing a curation method defined in this study. All the ARODs were successfully expressed in for analysis of their biochemical functions. The catalytic activity of our bacterially expressed ancestral ARODs suggests that our ASR was successful. The ancestral AROD that is phylogenetically most remote from a native AROD has the highest thermal stability and substrate promiscuity. Our findings led us to infer that AROD evolved from a highly thermostable and promiscuous LAAO. As an application, we can design artificial ARODs with improved functions compared with those of native ones.
追踪酶的进化轨迹有助于阐明酶在进化过程中如何获得其特征功能，如热适应和底物选择性。如果有足够的序列数据，祖先序列重建（ASR）是追踪进化过程的有效方法。通过 ASR 成功设计人工蛋白质需要从数据中选择序列来生成经过精心挑选的序列库。在这项研究中，我们试图追踪 l-精氨酸氧化酶（AROD）的进化轨迹，l-精氨酸氧化酶是一种依赖黄素腺嘌呤二核苷酸（FAD）的氨基酸氧化酶（LAAO），对 l-精氨酸具有很高的特异性。该库通过选择第 15、50、332 和 580 位残基分别为甘氨酸、丝氨酸、色氨酸和苏氨酸的序列来生成。我们排除了那些非常短或非常长以及序列同一性低的序列。使用该库设计了三个祖先 AROD（AncARODn0、AncARODn1 和 AncARODn2）。随后，我们在细菌中表达了祖先 AROD 以及天然 AROD（OkAROD）。AncARODn0 在系统发育上与 OkAROD 最远，而 AncARODn2 与 OkAROD 最相似。通过将 AROD 序列回溯到祖先进化，逐渐提高了热稳定性，而 AncARODn0 的残余活性达到最大活性的一半时的温度（）比 OkAROD 高>20°C。值得注意的是，只有 AncARODn0 表现出与传统的混杂性 LAAO 相似的广泛底物选择性。总的来说，我们的发现使我们推断 AROD 可能是从高度耐热和混杂性的 LAAO 进化而来的。在这项研究中，我们试图推断最近分离的依赖黄素腺嘌呤二核苷酸的 l-精氨酸氧化酶（AROD）的分子进化，该酶将 l-精氨酸氧化为 2-酮精氨酸。利用 10 个候选 AROD 序列，我们总共获得了三个祖先 AROD。此外，通过克隆候选 AROD 之一获得了一个天然 AROD。候选序列是使用本研究中定义的策方法选择的。所有 AROD 都在 中成功表达，以分析其生化功能。我们细菌表达的祖先 AROD 的催化活性表明我们的 ASR 是成功的。与天然 AROD 相比，在系统发育上最遥远的祖先 AROD 具有最高的热稳定性和底物混杂性。我们的发现使我们推断 AROD 是从高度耐热和混杂性的 LAAO 进化而来的。作为一种应用，我们可以设计具有比天然 AROD 更好功能的人工 AROD。
