Abstract
L-amino acid oxidases (LAAOs) can be applied to convert racemic amino acids to D-isomers, which are potential precursors of pharmaceuticals. However, this application is hampered by the lack of available stable and structure-determined LAAOs. In this study, we attempt to address this limitation by utilizing two ancestral LAAOs: AncLAAO-N4 and AncLAAO-N5. AncLAAO-N4 has the highest thermal and temporal stabilities among the designed LAAOs that can be used for deracemization and stereoinversion. AncLAAO-N5 can provide X-ray crystal structures, which are helpful to reveal substrate recognition and reaction mechanisms of LAAOs at the molecular level. Next, we attempted to improve activity of AncLAAO-N4 toward L-Val through a semi-rational protein engineering method. Three variants with enhanced activity toward L-Val were obtained. Taken together, we believe that the activity and substrate selectivity of AncLAAOs give them the potential to be key enzymes in various chemoenzymatic reactions.
Highlights
L-amino acid oxidases (LAAOs) can be applied to convert racemic amino acids to D-isomers, which are potential precursors of pharmaceuticals
Since D-amino acid oxidase (DAAO) have been used to synthesize various chemicals with chemoenzymatic reactions, the potential of LAAOs for application to this type of reaction has been of interest[22,42,43]
We designed two AncLAAOs, AncLAAO-N4 and AncLAAO-N5, which satisfy these conditions; AncLAAO-N4 is a highly stable LAAO which can be expressed in a heterologous expression system, and AncLAAO-N5 provides substrate recognition and the reaction mechanism of AncLAAOs at the molecular level
Summary
L-amino acid oxidases (LAAOs) can be applied to convert racemic amino acids to D-isomers, which are potential precursors of pharmaceuticals. Enzyme functions of DAAO were elucidated at the molecular level through multiple X-ray crystal structures[15,16] and biochemical analysis[17,18] These findings contribute to the design of DAAO variants bearing new substrate selectivity and high activity[14,19]. We designed an artificial LAAO (AncLAAON1 in this study, and AncLAAO in a previous study27) bearing broad substrate selectivity (>10 L-amino acids) and high productivity in an Escherichia coli expression system using ancestral sequence reconstruction (ASR). This enzyme can be applied to the deracemization of aromatic D-amino acids[27]. Low thermal stability and lack of experimental data for structural and functional analysis of AncLAAOs would prevent this application
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