Bioinformatics\nand Computationally Supported Redesign\nof Aspartase for β‑Alanine Synthesis by Acrylic Acid\nHydroamination

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