Improving the Catalytic Power of the DszD Enzyme for the Biodesulfurization of Crude Oil and Derivatives.

The enhancement of the catalytic power of enzymes is a subject of enormous interest both for science and for industry. The latter, in particular, due to the vast applications enzymes can have in industrial processes, for instance in the desulfurization of crude oil, which is mandatory by law in many developed countries and is currently performed using costly chemical processes. In this work we sought to enhance the turnover rate of DszD from Rhodococcus erythropolis, a NADH-FMN oxidoreductase responsible for supplying FMNH2 to DszA and DszC in the biodesulfurization process of crude oil, the 4S pathway. For this purpose, we replaced the wild type spectator residue of the rate limiting step of the reduction of FMN to FMNH2 , a process catalysed by DszD and known to play an important role in the reaction energy profile. As replacements, we used all the naturally occurring amino acids, one at a time, using computational methodologies, and repeated the above-mentioned reaction with each mutant. To calculate the different free energy profiles, one for each mutated model, we applied quantum mechanics/molecular mechanics (QM/MM) methods within an ONIOM scheme. The free energy barriers obtained varied between 15.1 and 29.9 kcal mol-1 . Multiple factors contributed to the different ΔG values. The most relevant were electrostatic interactions and the induction of a favourable alignment between substrate and cofactor. These results confirm the great potential that chirurgic mutations have for increasing the catalytic power of DszD in relation to the wild type (wt) enzyme.