Abstract
L-Amino acid oxidase (LAAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme active on most proteinogenic L-amino acids, catalysing their conversion to α-keto acids by oxidative deamination of the substrate. For this oxidation reaction, molecular oxygen is used as the electron acceptor, generating hydrogen peroxide. LAAO can be used to detect L-amino acids, for the production of hydrogen peroxide as an oxidative agent or antimicrobial agent, and for the production of enantiopure amino acids from racemates. In this work, we characterised a previously reported LAAO from the bacterium Pseudoalteromonas luteoviolacea. The substrate scope and kinetic properties of the enzyme were determined, and the thermostability was evaluated. Additionally, we elucidated the crystal structure of this bacterial LAAO, enabling us to test the role of active site residues concerning their function in catalysis. The obtained insights and ease of expression of this thermostable LAAO provides a solid basis for the development of engineered LAAO variants tuned for biosensing and/or biocatalysis.
Highlights
Introduction and Roland WohlgemuthL-Amino acid oxidase (LAAO) are flavin-dependent enzymes present in many organisms [1]
LAAOs catalyse the oxidative deamination of L-amino acids to α-keto acids, and substrate ranges have been determined for different LAAOs [5]
The UV–Vis absorbance spectrum of purified Pl-LAAO shows absorbance features that are typical for a flavincontaining protein, in line with its yellow appearance (Supplementary Figure S2)
Summary
Introduction and Roland WohlgemuthLAAOs are flavin-dependent enzymes present in many organisms [1]. The amino acid substrate is oxidised to the imino acid, which spontaneously reacts with water to form the α-keto acid and ammonium. This results in the formation of the flavin adenine dinucleotide (FAD) cofactor in the reduced state (FADH2 ). Regeneration of the reduced FAD by molecular oxygen leads to the production of hydrogen peroxide. It is the formation of this reactive oxygen species that has been the focus of studies concerning the role of LAAOs as inducers of oxidative stress [6]. The oxidative damage induced by LAAO activity from snake venom leads to cytotoxicity, antibacterial activity, and the disruption of platelet aggregation [7,8,9]
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