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 ( Hc LAAO4). Here, we crystallized a proteolytically truncated surface entropy reduction variant of Hc LAAO4 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 Hc LAAO4, 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.