Following the Evolutionary Track of a Highly Specific l-Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes

摘要: ABSTRACT Following the evolutionary track of enzymes can help elucidate how attain their characteristic functions, such as thermal adaptation and substrate selectivity, during process. Ancestral sequence reconstruction (ASR) is effective for following processes if sufficient data are available. Selecting sequences from to generate a curated library necessary successful design artificial proteins by ASR. In this study, we tried follow l-arginine oxidase (AROD), flavin adenine dinucleotide (FAD)-dependent amino acid (LAAO) that exhibits high specificity l-arginine. The was generated selecting in which 15th, 50th, 332nd, 580th residues Gly, Ser, Trp, Thr, respectively. We excluded either extremely short or long those with low degree identity. Three ancestral ARODs (AncARODn0, AncARODn1, AncARODn2) were designed using library. Subsequently, expressed well native Oceanobacter kriegii AROD (OkAROD) bacteria. AncARODn0 phylogenetically most remote OkAROD, whereas AncARODn2 similar OkAROD. Thermal stability gradually increased extending back progenitor, while temperature at residual activity half maximum measured ( T 1/2 ) >20°C higher than Remarkably, only exhibited broad selectivity conventional promiscuous LAAO. Taken together, our findings led us infer may have evolved highly thermostable IMPORTANCE attempted molecular evolution recently isolated FAD-dependent (AROD) oxidizes 2-ketoarginine. Utilizing 10 candidate sequences, obtained total three ARODs. addition, one cloning selected utilizing curation method defined study. All successfully Escherichia coli analysis biochemical functions. catalytic bacterially suggests ASR successful. has highest promiscuity. Our As an application, improved functions compared ones.