Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis.

The cytochrome P450 enzyme CYP121A1 endogenously catalyzes the formation of a carbon-carbon bond between the two phenol groups of dicyclotyrosine (cYY) in Mycobacterium tuberculosis (Mtb). One of 20 CYP enzymes in Mtb, CYP121A1 continues to garner significant interest as a potential drug target. The accompanying reports the use of 19F NMR spectroscopy, reconstituted activity assays, and molecular dynamics simulations to investigate the significance of hydrogen bonding interactions that were theorized to stabilize a static active site water network. The active site residue Asn-85, whose hydrogen bonds with the diketopiperazine ring of cYY contributes to a contiguous active site water network in the absence of cYY, was mutated to a serine (N85S) and to a glutamine (N85Q). These conservative changes in the hydrogen bond donor side chain result in inactivation of the enzyme. Moreover, the N85S mutation induces reverse type-I binding as measured by absorbance difference spectra. NMR spectra monitoring the ligand-adaptive FG-loop and the active site Trp-182 side chain confirm that disruption of the active site water network also significantly alters the structure of the active site. These data were consistent with dynamics simulations of N85S and N85Q that demonstrate that a compromised water network is responsible for remodeling of the active site B-helix and a repositioning of cYY toward the heme. These findings implicate a slowly exchanging water network as a critical factor in CYP121A1 function and a likely contributor to the unusual rigidity of the structure.

Active Site Aromatic Residues Play a Dual Role in the Substrate Interaction and Protein Structure in Functional Dimers of CYP121A1 of Mycobacterium tuberculosis.

The essential enzyme CYP121A1 of Mycobacterium tuberculosis forms a functional dimer, which when disrupted results in a decrease of activity and substrate specificity. The crystal structure of CYP121A1 in complex with its substrate di-cyclotyrosine (cYY) indicates that the aromatic side chains of Phe-168 and Trp-182 form stabilizing π-π interactions with a tyrosyl ring of cYY. In the enclosed study, we utilize targeted 19F labeling of aromatic residues to label CYP121A1 for detection by nuclear magnetic resonance (NMR) spectroscopy. 19F-NMR spectra and functional characterization of mutations to Phe-168 and Trp-182 are combined with all-atom molecular dynamics simulations of substrate-bound and substrate-free CYP121A1. This study shows that these aromatic residues interact with cYY predominantly through π-π stacking. In addition to playing an essential role in substrate binding, these active site residues also stabilize the tertiary and quaternary structures of CYP121A1. An additional unexpected finding was the presence of cYY-induced long-range allostery that affects residues located near the homodimer interface. Taken together, this study highlights a structural relationship between the active site environment of this essential enzyme with its global structure that was previously unknown.