title: Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of Rhodococcus erythropolis.
abstract: Microbial desulfurization is potentially an alternative process to chemical desulfurization of fossil fuels and their refined products. The dibenzothiophene desulfurizing system of Rhodococcus erythropolis includes DszD which is an NADH-dependent FMN oxidoreductase with 192 residues that is responsible for supplying reducing equivalents in the form of FMNH(2) to monooxygenases, DszA and DszC. We performed amino acid sequence comparisons and structural predictions based on the crystal structure of available pdb files for three flavin reductases PheA2, HpaC(Tt) and HpaC(St) with the closest structural homology to IGTS8 DszD. The Thr62 residue in DszD was substituted with Asn and Ala by site-directed single amino acid mutagenesis. Variants T62N and T62A showed 5 and 7 fold increase in activities based on the recombinant wild type DszD, respectively. This study revealed the critical role of position 62 in enzyme activity. These results represent the first experimental report on flavin reductase mutation in R. erythropolis and will pave the way for further optimization of the biodesulfurization process.
authors.affiliation: National Institute for Genetic Engineering and Biotechnology (NIGEB), 14178, Tehran, Iran.
authors.forename: Nasrin
authors.initials: N
authors.lastname: Kamali
authors.forename: Mahmood
authors.initials: M
authors.lastname: Tavallaie
authors.forename: Bijan
authors.initials: B
authors.lastname: Bambai
authors.forename: Ali Asghar
authors.initials: AA
authors.lastname: Karkhane
authors.forename: Mandana
authors.initials: M
authors.lastname: Miri
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D038181:FMN Reductase / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D038181:FMN Reductase