ABSTRACT
l-threonine aldolase (LTA) catalyzes the synthesis of ß-hydroxy-α-amino acids, which are important chiral intermediates widely used in the fields of pharmaceuticals and pesticides. However, the limited thermostability of LTA hinders its industrial application. Furthermore, the trade-off between thermostability and activity presents a challenge in the thermostability engineering of this enzyme. This study proposes a strategy to regulate the rigidity of LTA's V-shaped subunit by modifying its opening and hinge regions, distant from the active center, aiming to mitigate the trade-off. With LTA from Bacillus nealsonii as targeted enzyme, a total of 25 residues in these two regions were investigated by directed evolution. Finally, mutant G85A/M207L/A12C was obtained, showing significantly enhanced thermostability with a 20 °C increase in T5060 to 66 °C, and specific activity elevated by 34 % at the optimum temperature. Molecular dynamics simulations showed that the newly formed hydrophobicity and hydrogen bonds improved the thermostability and boosted proton transfer efficiency. This work enhances the thermostability of LTA while preventing the loss of activity. It opens new avenues for the thermostability engineering of other industrially relevant enzymes with active center located at the interface of subunits or domains.
Subject(s)
Enzyme Stability , Molecular Dynamics Simulation , Mutation , Temperature , Bacillus/enzymology , Bacillus/genetics , Hydrogen Bonding , Aldehyde-Lyases/chemistry , Aldehyde-Lyases/genetics , Aldehyde-Lyases/metabolism , Catalytic Domain , Kinetics , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Protein Engineering/methodsABSTRACT
l-threonine aldolase (LTA) catalyzes C-C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active-site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 %syn to 99.4 %syn . Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 %anti . Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C-C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C-C bond-forming enzymes.
Subject(s)
Amino Acids , Glycine Hydroxymethyltransferase , Glycine Hydroxymethyltransferase/genetics , Glycine Hydroxymethyltransferase/metabolism , Mutation , Mutagenesis , Amino Acids/chemistry , Catalytic Domain , Substrate SpecificityABSTRACT
The L-threonine aldolase from Leishmania major was engineered to improve its diastereoselectivity by a CAST/ISM strategy, providing insights into the relationship between the physico-chemical properties of the substrate access path and diastereoselectivity. The steric hindrance, hydrophobic interaction and π-π interaction cooperated to improve the diastereoselectivity of the enzyme, with a diastereomeric excess (de) value reaching 96.3%syn from 26.8%syn.