Computational design of the temperature optimum of an enzyme reaction

• Published in: Science Advances
• Main Authors: van der Ent, Florian, Skagseth, Susann, Lund, Bjarte A., Sočan, Jaka, Griese, Julia J., Brandsdal, Bjørn O., Åqvist, Johan
• Format: Text
• Language: English
• Published: American Association for the Advancement of Science 2023
• Subjects: Physical and Materials Sciences; Antarctic; Antarc*
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10306287/; http://www.ncbi.nlm.nih.gov/pubmed/37379391; https://doi.org/10.1126/sciadv.adi0963

Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog.