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

Abstract

l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination of l-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 basic l-amino acids. In this study, we employed a LAAD from Proteus 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 substrate l-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward various l-amino acid substrates. The variant PmiLAADE417K showed remarkable catalytic activity improvement on seven substrates (l-Ala, l-Asp, l-Ile, l-Leu, l-Phe, l-Trp, and l-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.

Mechanism-Guided Computational Design Drives meso-Diaminopimelate Dehydrogenase to Efficient Synthesis of Aromatic d-amino Acids

“…Several biological methods have been developed for producing aromatic d -AAs, including hydrolysis, − chiral resolution, − and asymmetric catalysis. − Hydrolysis involves the transformation of a racemic substrate (including d -amino acid amides or peptides comprising d -amino acids) into a d -amino acid with stereospecific hydrolases. This includes N -acyl- d -amino acid amide hydrolase, d -amino acid amidase, or alkaline d -peptidase. − Chiral resolution is the use of stereospecific enzymes, such as l -amino acid deaminase, l -ammonia lyase, or l -amino acid oxidase − , to catalyze the conversion of l -amino acids into keto acid, thus achieving the chiral resolution of racemic amino acids into aromatic d -AAs.…”