Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination ofl-amino acids into α-keto acids, which are widely used in the pharmaceutical, food, chemical, and cosmetic industries. However, the substrate specificity of available LAADs is limited, and most substrates are concentrated on several bulky or basicl-amino acids. In this study, we employed a LAAD fromProteus mirabilis(PmiLAAD) and broadened its substrate specificity using a semi-rational design strategy. Molecular docking and alanine scanning identified F96, Q278, and E417 as key residues around the substrate-binding pocket of PmiLAAD. Site-directed saturation mutagenesis identified E417 as the key site for substrate specificity expansion. Expansion of the substrate channel with mutations of E417 (E417L, E417A) improved activity toward the bulky substratel-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward variousl-amino acid substrates. The variant PmiLAADE417Kshowed remarkable catalytic activity improvement on seven substrates (l-Ala,l-Asp,l-Ile,l-Leu,l-Phe,l-Trp, andl-Val). The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions. These PmiLAAD variants with a broadened substrate spectrum can extend the application potential of LAADs.Keywords:l-amino deaminase;amino acids;keto acids;substrate spectrum;substrate channel