Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations
| Autor: | Manon Couture, Normand Cyr, Julie Barma, Mahder Seifu Manenda, John M. Pascal, Xiaojun Zhu, Rong Shi, Changsheng Zhang, Li-Ping Zhang, Marie-Ève Picard |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Stereochemistry Pyridines Flavoprotein Flavin group Random hexamer Molecular Dynamics Simulation Crystallography X-Ray Biochemistry Cofactor Mixed Function Oxygenases Substrate Specificity 03 medical and health sciences Bacterial Proteins X-Ray Diffraction Scattering Small Angle Molecular Biology Binding Sites 030102 biochemistry & molecular biology biology Futile cycle Chemistry Cell Biology NADPH oxidation Monooxygenase Streptomyces Protein Structure Tertiary 030104 developmental biology Protein Structure and Folding biology.protein Biocatalysis Flavin-Adenine Dinucleotide Mutagenesis Site-Directed NADPH binding Protein Multimerization Oxidation-Reduction NADP Protein Binding |
| Zdroj: | J Biol Chem |
| ISSN: | 1083-351X |
| Popis: | Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen-oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a "sliding" conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes. |
| Databáze: | OpenAIRE |
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