Abstract
Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.
| Original language | English |
|---|---|
| Pages (from-to) | 915-923 |
| Number of pages | 9 |
| Journal | Biotechnology and Applied Biochemistry |
| Volume | 66 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 1 Nov 2019 |
Bibliographical note
Publisher Copyright:© 2019 International Union of Biochemistry and Molecular Biology, Inc.
Funding
This work was supported partially by a TUBITAK grant (110M001), and funds from Istanbul Technical University Scientific Project Support Center. G.G. and R.I. carried out all the cloning and purification experiments of SSO2055 and participated in drafting the manuscript. O.T. and Y.Y. analyzed the homology model and protein structure and contributed to the draft. A.T.B. performed LC–MS/MS experiments. G.D.D. and Y.Y. conceived of the study and participated in its design and coordination. G.D.D. resolved final approval of the version to be published. All authors read and approved the final manuscript. This work was supported partially by a TUBITAK grant (110M001), and funds from Istanbul Technical University Scientific Project Support Center. G.G. and R.I. carried out all the cloning and purification experiments of SSO2055 and participated in drafting the manuscript. O.T. and Y.Y. analyzed the homology model and protein structure and contributed to the draft. A.T.B. performed LC?MS/MS experiments. G.D.D. and Y.Y. conceived of the study and participated in its design and coordination. G.D.D. resolved final approval of the version to be published. All authors read and approved the final manuscript.
| Funders | Funder number |
|---|---|
| TUBITAK | 110M001 |
| Istanbul Teknik Üniversitesi | SSO2055 |
Keywords
- archaea
- biodesulfurization
- flavin reductase activity
- hyperthermophile
- oxidoreductase