8RJ0

Crystal structure of mutant aspartase from Bacillus sp. YM55-1 in the closed loop conformation

Summary for 8RJ0

• Entry DOI: 10.2210/pdb8rj0/pdb
• Descriptor: Aspartate ammonia-lyase, TRIETHYLENE GLYCOL, SODIUM ION, ... (4 entities in total)
• Functional Keywords: ammonia aspartate lyase, closed conformation, lyase
• Biological source: Bacillus sp. YM55-1
• Total number of polymer chains: 2
• Total formula weight: 103492.07

Authors

Capra, N.,Thunnissen, A.M.W.H.,Janssen, D.B. (deposition date: 2023-12-19, release date: 2025-01-15, Last modification date: 2025-02-05)

Primary citation

Gran-Scheuch, A.,Wijma, H.J.,Capra, N.,van Beek, H.L.,Trajkovic, M.,Baldenius, K.,Breuer, M.,Thunnissen, A.W.H.,Janssen, D.B.
Bioinformatics and Computationally Supported Redesign of Aspartase for beta-Alanine Synthesis by Acrylic Acid Hydroamination.
Acs Catalysis, 15:928-938, 2025

PubMed Abstract: Aspartate ammonia lyases catalyze the reversible amination of fumarate to l-aspartate. Recent studies demonstrate that the thermostable enzyme from 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 values with acrylic acid of at least 0.6-1.5 s with values in the high mM range. Since the β-alanine production of wild-type enzyme was below the detection limit, this suggests that the / was improved by at least 1000-fold. Crystal structures of the 6-fold mutant of redesigned AspB and the similarly engineered aspartase from 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.

PubMed: 39839848
DOI: 10.1021/acscatal.4c05525

Experimental method

X-RAY DIFFRACTION (1.9 Å)