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 <i>Bacillus</i> 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 <i>k</i> _cat values with acrylic acid of at least 0.6-1.5 s^-1 with <i>K</i> _M values in the high mM range. Since the β-alanine production of wild-type enzyme was below the detection limit, this suggests that the <i>k</i> _cat/<i>K</i> _m was improved by at least 1000-fold. Crystal structures of the 6-fold mutant of redesigned AspB and the similarly engineered aspartase from <i>Caenibacillus caldisaponilyticus</i> 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.