19F‐NMR shows that active site aromatic residues in CYP121 of Mycobacterial tuberculosisplay a dual role in substrate interaction and protein structure

Abstract

CYP121 is an essential cytochrome P450 enzyme (CYP) from Mycobacterium tuberculosis.CYP121 mediates an atypical phenol‐coupling reaction that converts cyclodityrosine (cYY) into mycocylosin. Our lab has previously employed a 19F nuclear magnetic resonance (NMR) label, 3‐bromo‐1,1,1‐trifluoroacetone (BTFA), to study dynamics of the FG‐loop, a region connecting the F and G‐helices. In other CYPs, the FG‐loop region has been implicated in active site access and substrate recognition. The 19F‐NMR spectrum for the FG‐loop in CYP121 is heterogeneous, suggesting that in solution there are multiple conformations. Upon titration of cYY, the signal converges to a single peak representing a bound state. Interestingly, this labeling site exists approximately 20 angstroms away from the active site heme, where ligand binding is typically measured in CYPs by UV‐visible spectroscopy. Crystal structures show an invariable protein structure with ligands clustered near the FG‐helices, away from the heme.Here we investigate the specificity of this interaction site by expanding our use of 19F labeling. Supplementing cells with 5‐fluoroindole (5‐FI) during recombinant protein expression, 19F was incorporated into each tryptophan residue of CYP121. This method of labeling allowed for four tryptophan residues to be monitored, which span the entire protein structure. Notably, two residues, Trp‐182 and Trp‐380, are located adjacent to the CYP121 homodimer interface. The 19F‐NMR spectrum contains three distinct signals, two of which are equal in intensity, and a third which has been determined to be two overlapping resonances. Addition of cYY causes a shift of equilibrium in the spectra, with the overlapped resonances resolving and resulting in four peaks of roughly equal area and intensity. These results suggest that only the active site residue Trp‐182 is affected by the addition of cYY. Using this labeling scheme, we also tested alanine substitutions of Trp‐182 and another active site aromatic residue, Phe‐168. 19F‐NMR spectra from these mutants indicate a general perturbation of the structure, with only one Trp residue left unaffected. Since Trp‐182 extends into a hydrophobic pocket of CYP121 (suggesting a structural role), additional substitutions were made using tyrosine and leucine side chains. The conservative W182Y mutant yielded similar disorder as seen when replaced with alanine, while W182L results a partially restored spectrum. Interestingly, both mutants bind substrate in a similar manner as wild‐type when measured by UV‐visible spectroscopy, although with weakened affinity.These findings indicate that while Phe‐168 and Trp‐182 are involved in substrate interaction, they also play a role in the fold of the protein, with mutagenesis causing long range changes in structure.