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

Abstract

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 Oceanobacter kriegii 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 (T1/2) 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. IMPORTANCE 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 Escherichia coli 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.

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

“…Therefore, ASR is currently being adopted as a tool for protein engineering (25). For example, new LAAOs which bear broad substrate selectivity (> 10 Lamino acids) could be assigned by a paralog search of L-arginine oxidase (AROD) (26)(27)(28). Artificial LAAOs can be designed by ASR utilizing six of the assigned LAAOs as a sequence library.…”

“…On the other hand, there are thousands of protein sequences belonging to the LAAO superfamily in databases (29), and the currently available functionally characterized sequences are only the tip of the iceberg. Several sequences, such as AROD (27,30), AncLAAO (26,28), and Ltryptophan oxidase (VioA) (31)(32)(33), can now be functionally annotated. Thus, there is an opportunity to acquire new LAAOs from the database using in silico enzyme screening methods.…”

“…In this study, we attempted to assign new LAAOs from the database utilizing one of the previously designed AncLAAO sequences (28) as a template. Through combinational approaches using paralog searches, the application of previously reported original protein sequence selection methods (27,(34)(35)(36), and biochemical assays, we newly characterized LAAOs exhibiting high specificity toward L-Lys. These included L-Lysine oxidase (LLysO) from the Chryseobacterium, Flavobacterium, and Pedobacter species.…”

Faculty Opinions recommendation of AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.

“…A comprehensive and curated sequence library was prepared querying the Blastp web server and using a custom Python script (Source code 1), which exhibited more than 30% sequence identity with E. coli BirA (EU08004.1). Next, further curation approaches were applied to the library as shown in previous studies (Nakano et al, 2018;Nakano et al, 2019). The procedure consists of the following steps: 1) preparation of sequence pairs consisting of one of the submitted BirA sequences and one sequence (total 1275 genes) in the library, 2) sequence alignment of the all pairs, and 3) selection of sequences bearing 'key residues' Figure 1.…”

“…Evolutionary protein engineering using metagenome data have recently been used to improve enzymes (Nakano and Asano, 2015;Nakano et al, 2018;Nakano et al, 2019). Here, we newly designed five ancestral BirA enzymes using an ancestral enzyme reconstruction algorithm and a large genome dataset.…”