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

Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a “ninja-star” feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger k cat / K m values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in k cat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac -forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs. D-Amino acids are widely present in nature and display diverse physiological functions, however, their large-scale synthesis remains challenging for specific amino acids. Here, the authors design hyper-thermostable ancestral L-amino acid oxidases (HTAncLAAO2), catalyzing the chemoenzymatic synthesis of D-tryptophan from L-tryptophan at a preparative scale.