Simulation-Based protein engineering of R. erythropolis NADH-FMN Oxidoreductase

Abstract

The sulfur contents of fossil fuels has negative impacts on the environment and human health. The bio-catalytic desulfurization strategies and the biological refinement of the fossil fuels are a cost-effective process compared to chemical desulfurization. Rhodococcus erythropolis IGTS8 is able to extract the organic sulfur of oil as mineral salts by 4S metabolic pathway (i.e. DszA,B,C and D genes). dszD gene codes a NADH:FMN oxidoreductase delivering reducing equivalent to DszA, DszB, and DszC to remove sulfur from heterocyclic molecules. In this study, we sought to improve DszD specificity by side-direct mutagenesis based on prediction of the DszD structure, molecular docking, and molecular dynamic simulation. Accordingly, the cloning, expression and activity assay of best candidates (A79I and A79N) were performed. Our results demonstrated the important role of position 79 in enzyme activity, the A79I and A79N mutants are able to increase the enzyme activity 3.4 and 5.2 fold compared to wild-type.