Abstract

Life could not be sustained without the presence of enzymes, which are responsible for accelerating the chemical reactions in a biologically compatible timescale. Enzymes present additional advantageous features such as high specificity and selectivity, plus they operate under very mild biological conditions. Inspired by these extraordinary characteristics, many scientists wondered about the possibility of designing new enzymes for industrially-relevant targets. Enzymes exist as an ensemble of conformational states, whose populations can be shifted by substrate binding, allosteric interactions, but also by introducing mutations to their sequence. Tuning the populations of the enzyme conformational states through mutation enables evolution towards novel activity.[1] A common feature observed in many laboratory-evolved enzymes, is the introduction of remote mutations from the catalytic center, which often have a profound effect in the enzyme catalytic activity. [2] As it happens in allosterically regulated enzymes, distal mutations regulate the enzyme activity by stabilizing pre-existing catalytically important conformational states. In this talk, the rational enzyme design approaches our group has developed based on inter-residue correlations from microsecond time-scale Molecular Dynamics (MD) simulations and enhanced sampling techniques will be presented. [3,4] Our work along the years in many different enzyme systems evidences that the current challenge of distal active site prediction for enhanced function in computational enzyme design can be ultimately addressed.[3] References: [1] Maria-Solano, M. A.; Serrano-Hervás, E.; Romero-Rivera, A.; Iglesias-Fernández, J.; Osuna, S. Role of conformational dynamics for the evolution of novel enzyme function, Chem. Commun. 2018, 54, 6622-6634. [2] Osuna, S. The challenge of predicting distal active site mutations in computational enzyme design, WIREs Comput Mol Sci. 2020, e1502. [3] Maria-Solano, M. A.; Kinateder, T.; Iglesias-Fernández, J.; Sterner, R.; Osuna, S. In Silico Identification and Experimental Validation of Distal Activity-Enhancing Mutations in Tryptophan Synthase, ACS Catal. 2021, 11, 13733-13743. [4] Casadevall, G.; Duran, C.; Estévez-Gay, M.; Osuna, S. Estimating conformational heterogeneity of tryptophan synthase with a template-based AlphaFold2 approach, Prot. Sci. 2022, 31, e4426.