Reaction Mechanism of Ancestral l-Lys α-Oxidase from Caulobacter Species Studied by Biochemical, Structural, and Computational Analysis

Abstract

The flavin-dependent amine oxidase superfamily contains various l-amino acid oxidases (LAAOs) bearing different substrate specificities and enzymatic properties. LAAOs catalyze the oxidation of the α-amino group of l-amino acids (L-AAs) to produce imino acids and H2O2. In this study, an ancestral l-Lys α-oxidase (AncLLysO2) was designed utilizing genome-mined sequences from the Caulobacter species. The AncLLysO2 exhibited high specificity toward l-Lys; the k cat/K m values toward l-Lys were one and two orders larger than those of l-Arg and l-ornithine, respectively. Liquid chromatography–high resolution mass spectrometry analysis indicated that AncLLysO2 released imino acid immediately from the active site after completion of oxidation of the α-amino group. Crystal structures of the ligand-free, l-Lys- and l-Arg-bound forms of AncLLysO2 were determined at 1.4–1.6 Å resolution, indicating that the active site of AncLLysO2 kept an open state during the reaction and more likely to release products. The structures also indicated the substrate recognition mechanism of AncLLysO2; ε-amino, α-amino, and carboxyl groups of l-Lys formed interactions with Q357, A551, and R77, respectively. Biochemical and molecular dynamics simulation analysis of AncLLysO2 indicated that active site residues that indirectly interact with the substrate are also important to exhibit high activity; for example, the aromatic group of Y219 is important to ensure that the l-Lys substrate is placed in the correct position to allow the reaction to proceed efficiently. Taken together, we propose the reaction mechanism of AncLLysO2.

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction

“…HTAncLAAO2 bears a high sequence identity with the HTAncLAAO 27 , suggesting that substrate recognition mechanisms would be conserved to each other. The overall structure of HTAncLAAO2, determined at a 2.2 Å resolution (Table S4 ), indicating that the enzyme has typical folding of amine oxidase superfamily, such as L-Lysine α-oxidase 34 , 35 and LAAO from Pseudoalteromonas species 26 , 36 , in spite that they shared low sequence identity to each other. The unique point of HTAncLAAO2 is that the enzyme displays a distinctive octameric state (Fig.…”

Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

“…ASR, a sequence-based protein redesign approach, reconstructs ancestral sequences using provided multiple sequence alignment and phylogenetic tree data . These ancestral sequences often have properties that are suitable for the applications, such as increased thermostability, solubility, new activity, and broader substrate selectivity. − ASR can also be used to identify enzymes with specific desired characteristics by altering input data. − Through this screening and reconstruction process, we identified a novel ancestral SrtE variant, termed AcSE5. In this study, we elucidate the biochemical properties of AcSE5, demonstrating its suitability for producing protein conjugates and immobilized enzymes.…”