19F-NMR reveals substrate specificity of CYP121A1 in Mycobacterium tuberculosis
| Autor: | D. Fernando Estrada, George E. Ghanatios, Christopher S. Campomizzi |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
TB
tuberculosis Stereochemistry Dimer Mtb Mycobacterium tuberculosis Cytochrome P450 Fluorine-19 NMR DMSO dimethyl sulfoxide Biochemistry chemistry.chemical_compound CYP121A1 Side chain CYPs cytochrome P450 Molecular Biology 19F fluorine chemistry.chemical_classification Dipeptide biology Substrate (chemistry) Cell Biology NMR Enzyme chemistry biology.protein BTFA 3-bromo-1 1 1-trifluoroacetone cYY dicyclotyrosine Function (biology) Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/j.jbc.2021.101287 |
| Popis: | Cytochromes P450 are versatile enzymes that function in endobiotic and xenobiotic metabolism and undergo meaningful structural changes that relate to their function. However, the way in which conformational changes inform the specific recognition of the substrate is often unknown. Here, we demonstrate the utility of fluorine (19F)-NMR spectroscopy to monitor structural changes in CYP121A1, an essential enzyme from Mycobacterium tuberculosis. CYP121A1 forms functional dimers that catalyze the phenol-coupling reaction of the dipeptide dicyclotyrosine. The thiol-reactive compound 3-bromo-1,1,1-trifluoroacetone was used to label an S171C mutation of the enzyme FG loop, which is located adjacent to the homodimer interface. Substrate titrations and inhibitor-bound 19F-NMR spectra indicate that ligand binding reduces conformational heterogeneity at the FG loop in both the dimer and in an engineered monomer of CYP121A1. However, only the dimer was found to promote a substrate-bound conformation that was preexisting in the substrate-free spectra, thus confirming a role for the dimer interface in dicyclotyrosine recognition. Moreover, 19F-NMR spectra in the presence of substrate analogs indicate the hydrogen-bonding feature of the dipeptide aromatic side chain as a dicyclotyrosine specificity criterion. This study demonstrates the utility of 19F-NMR as applied to a multimeric cytochrome P450, while also revealing mechanistic insights for an essential M. tuberculosis enzyme. |
| Databáze: | OpenAIRE |
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