Improvement of the activity and thermostability of l-threonine aldolase from Pseudomonas putida via tailoring of the active sites lining the binding pocket

l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesize β-hydroxy-α-amino acids with unsatisfactory enzyme activity. Here, we expressed the l-threonine aldolase from Pseudomonas putida KT2440 (l-PpTA) in Escherichia coli BL21 (DE3) and improved the activity and thermostability by protein engineering. Five amino acid residues (Ser10, His89, Asp93, Arg177, and Arg321) located in the substrate-binding pocket were selected and for mutation. Eight mutants (D93A, D93G, D93M, D93F, D93S, D93Q, D93Y and D93H) with increased enzyme activity were identified and their kcat/KM values showed about 1–7-fold higher than wild-type. Among all the variants, D93H showed the highest catalytic efficiency with 2925 and 4515 s−1 mM−1 of kcat/KM values toward l-threonine and l-allo-threonine, respectively. In addition, circular dichroism spectrum exhibited that the melting temperature of D93H (54.2 °C) was 5 °C higher than wild-type (49.2 °C). Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate, which facilitated the proton extraction and promote the enzymatic reaction. This work affords a candidate for the synthesis of β-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.