Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of Rhodococcus erythropolis

• Published in: Biotechnology letters Vol. 32; no. 7; pp. 921 - 927
• Main Authors: Kamali, Nasrin, Tavallaie, Mahmood, Bambai, Bijan, Karkhane, Ali Asghar, Miri, Mandana
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.07.2010; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Amino acids; Applied Microbiology; Biochemistry; Biodesulfurization; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Desulfurizing; DszD; FMN oxidoreductase; FMN Reductase - genetics; FMN Reductase - metabolism; Fundamental and applied biological sciences. Psychology; Life Sciences; Microbiology; Microorganisms; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Original Research Paper; Oxidation-Reduction; Protein Structure, Tertiary; Recombinant; Reductases; Residues; Rhodococcus; Rhodococcus - enzymology; Rhodococcus - genetics; Rhodococcus erythropolis; Rhodococcus erythropolis IGTS8; Sequence Alignment; site-directed mutagenesis; Sulfur content; Supplying; DszD; Biodesulfurization; IGTS8; FMN oxidoreductase; Site-directed mutagenesis; Desulfurization; Enzyme; Site directed mutagenesis; Rhodococcus erythropolis; Rhodococcus erythropolis IGTS8; NADH; Bacteria; Actinomycetes; Oxidoreductases
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-010-0254-4

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 FMNH₂ 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, HpaCTt and HpaCSt 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.