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

Sohei Ito,Sayaka Sugiura,Shogo Nakano,Masazumi Niwa,Fumihito Hasebe,Daisuke Matsui

doi:10.1016/j.jbc.2021.101043

Abstract

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.

Highlights

Expressed in many species, from bacteria to mammals [6,7,8,9,10]
We reported that paralog search and sequence library classification using several key residues as a motif are effective for finding new enzymes from sequence databases bearing unique properties, such as broad substrate selectivity and high thermostability [26, 35, 36]
In the ligand-free form, a cavity at the active site to recognize substrates is occupied by the side-chain Y254, which is the residue forming the plug loop (A in Fig. 7); this occupation would bring about structural changes of the substrate recognition residues, such as E383 and Y516 and made it difficult to predict the recognition mechanism solely from the structure of the AncLLysO ligandfree form

Summary

Introduction

Expressed in many species, from bacteria to mammals [6,7,8,9,10]. LAAO catalyzes the oxidation of the main chain amino group of L-amino acids and produces imino acids; these products are released into solvents and are quickly hydrolyzed to keto acid [4]. LAAOs bearing high substrate specificity could be used to quantify specific L-amino acid concentrations in various samples These LAAOs are named based on their substrate specificity and are the main focus of this study. New LAAOs that bear broad substrate selectivity (>10 L-amino acids) could be assigned by a paralog search of L-arginine oxidase (AROD) [26,27,28]. The designed enzyme, called AncLAAO, could be produced using the Escherichia coli expression system with the highest yield among any previously reported LAAOs (>50 mg/l) These can be applied to Structural and functional analysis of AncLLysO deracemize dozens of racemic amino acid derivatives to their Dforms with high enantiopurity [26]. The combination of biochemical and structural analysis of AncLLysO and its variants revealed the substrate recognition and reaction mechanism of LLysO at a molecular level

Results

Discussion

Conclusion