biostudies-literatureXue JMinistry of Science and Technology of the People's Republic of ChinaNational Natural Science Foundation of China13868-13877https://www.ebi.ac.uk/biostudies/studies/S-EPMC9064048biostudies-literatureUnknown9(24)The poor predictive accuracy of current computational enzyme design methods has led to low success rates of producing highly active variants that target non-natural substrates. In this report, a quantitative assessment approach based on molecular dynamics (MD) simulations was developed to eliminate false-positive enzyme designs at the computational stage. Taking cephradine hydrolase as an example, the apparent Michaelis binding constant (K m) and catalytic efficiency (k cat/K m) of designed variants were correlated with binding free energies and activation energy barriers, respectively, as calculated by molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics/generalized Born surface area (MM/GBSA) methods wiRSC advancesUsing molecular dynamics simulations to evaluate active designs of cephradine hydrolase by molecular mechanics/Poisson-Boltzmann surface area and molecular mechanics/generalized Born surface area methods.PMC9064048214761232012AA02120421878170Zhu YHuang XXue JfalseUsing molecular dynamics simulations to evaluate active designs of cephradine hydrolase by molecular mechanics/Poisson-Boltzmann surface area and molecular mechanics/generalized Born surface area methods.The poor predictive accuracy of current computational enzyme design methods has led to low success rates of producing highly active variants that target non-natural substrates. In this report, a quantitative assessment approach based on molecular dynamics (MD) simulations was developed to eliminate false-positive enzyme designs at the computational stage. Taking cephradine hydrolase as an example, the apparent Michaelis binding constant (K m) and catalytic efficiency (k cat/K m) of designed variants were correlated with binding free energies and activation energy barriers, respectively, as calculated by molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics/generalized Born surface area (MM/GBSA) methods wi2019-01-01T00:00:00Z2019 Apr2026-05-30T15:17:31.047Z2025-04-04T10:01:57.53ZS-EPMC90640483551954310.1039/c9ra02406a