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¹⁹F-NMR reveals substrate specificity of CYP121A1 in Mycobacterium tuberculosis.

ABSTRACT: Cytochromes P450 (CYPs) 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 substrate is often unknown. Here we demonstrate the utility of ¹⁹F nuclear magnetic resonance (NMR) spectroscopy to monitor structural changes in CYP121A1, an essential enzyme from Mycobacterium tuberculosis (Mtb). CYP121A1 forms functional dimers that catalyze the phenol-coupling reaction of the dipeptide dicylcotyrosine (cYY). The thiol-reactive compound 3-bromo-1,1,1-trifluoroacetone (BTFA) 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 pre-existing in the substrate-free spectra, thus confirming a role for the dimer interface in cYY recognition. Moreover, ¹⁹F-NMR spectra in the presence of substrate analogs indicate the hydrogen-bonding feature of the dipeptide aromatic side chain as a strict specificity criterion. This study demonstrates the utility of ¹⁹F-NMR as applied to a multimeric CYP, while also revealing mechanistic insight for an essential Mtb enzyme.

SUBMITTER: Campomizzi CS

PROVIDER: S-EPMC8571521 | biostudies-literature |

REPOSITORIES: biostudies-literature

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