Bioinformatics and Computationally Supported Redesign of Aspartase for β-Alanine Synthesis by Acrylic Acid Hydroamination

Aspartate ammonia lyases catalyze the reversible amination of fumarate to l-aspartate. Recent studies demonstrate that the thermostable enzyme from Bacillus sp. YM55–1 (AspB) can be engineered for the enantioselective production of substituted β-amino acids. This reaction would be attractive for the conversion of acrylic acid to β-alanine, which is an important building block for the preparation of bioactive compounds. Here we describe a bioinformatics and computational approach aimed at introducing the β-alanine synthesis activity. Three strategies were used: First, we redesigned the α-carboxylate binding pocket of AspB to introduce activity with the acrylic acid. Next, different template enzymes were identified by genome mining, equipped with a redesigned α-carboxylate pocket, and investigated for β-alanine synthesis, which yielded variants with better activity. Third, interactions of the SS-loop that covers the active site and harbors a catalytic serine were computationally redesigned using energy calculations to stabilize reactive conformations and thereby further increase the desired β-alanine synthesis activity. Different improved enzymes were obtained and the best variants showed kcat values with acrylic acid of at least 0.6–1.5 s–1 with KM values in the high mM range. Since the β-alanine production of wild-type enzyme was below the detection limit, this suggests that the kcat/Km was improved by at least 1000-fold. Crystal structures of the 6-fold mutant of redesigned AspB and the similarly engineered aspartase from Caenibacillus caldisaponilyticus revealed that their ligand-free structures have the SS-loop in a closed (reactive) conformation, which for wild-type AspB is only observed in the substrate-bound enzyme. AlphaFold-generated models suggest that other aspartase variants redesigned for acrylic acid hydroamination also prefer a 3D structure with the loop in a closed conformation. The combination of binding pocket redesign, genome mining, and enhanced active-site loop closure thus created effective β-alanine synthesizing variants of aspartase.