Mutability-Landscape-Guided Engineering of l-Threonine Aldolase Revealing the Prelog Rule in Mediating Diastereoselectivity of C-C Bond Formation

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.

Automated summary

In this study, the diastereoselectivity of L-threonine aldolase was tuned using mutagenesis and iterative saturation mutation strategies. The molecular dynamics simulations and coevolution analysis provided insights into the diastereoselectivity regulation mechanism. This research is valuable for expanding the applications of L-threonine aldolase and guiding the engineering of other C-C bond-forming enzymes.