RESUMO
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.
Assuntos
Aminoácidos , Glicina Hidroximetiltransferase , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Mutação , Mutagênese , Aminoácidos/química , Domínio Catalítico , Especificidade por SubstratoRESUMO
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.