Crystal structure and enzyme engineering of the broad substrate spectrum l‐amino acid oxidase 4 from the fungus Hebeloma cylindrosporum

l‐Amino acid oxidases (LAAOs) catalyze the oxidative deamination of l‐amino acids to α‐keto acids. Recombinant production of LAAOs with broad substrate spectrum remains a formidable challenge. We previously achieved this for the highly active and thermostable LAAO4 of Hebeloma cylindrosporum (HcLAAO4). Here, we crystallized a proteolytically truncated surface entropy reduction variant of HcLAAO4 and solved its structure in substrate‐free form and in complex with diverse substrates. The ability to support the aliphatic portion of a substrate's side chain by an overall hydrophobic active site is responsible for the broad substrate spectrum of HcLAAO4, including l‐amino acids with big aromatic, acidic and basic side chains. Based on the structural findings, we generated an E288H variant with increased activity toward pharmaceutical building blocks of high interest. l‐amino acid oxidases (LAAO) catalyze the oxidative deamination of l‐amino acids to α‐keto acids. The availability of LAAOs suitable for biotechnological applications is limited. Here, we crystallized the highly active LAAO4 from Hebeloma cylindrosporum and solved its structure in substrate‐free form and in complex with diverse substrates. A designed variant with increased activity toward pharmaceutical building blocks of high interest was generated.