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

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the kcat/Km value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.

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

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.