Abstract 1168 Water as structure in a cytochrome P450 enzyme: CYP121 from Mycobacterium tuberculosis relies on an ordered active site water network for its structure and function

The cytochrome P450 enzyme CYP121 forms functional dimers that catalyze formation of a carbon-carbon bond between the two phenol groups of dicyclotyrosine (cYY) in Mycobacterium tuberculosis (Mtb). One of twenty CYP enzymes in Mtb, the essentiality of CYP121 and the unusual nature of its catalytic reaction means that it continues to garner significant interest as a potential drug target. The objective of this study was to investigate the structural and functional role of the active site water network in CYP121. We relied on mutagenesis of active site residues, 19F-NMR spectroscopy, reconstituted activity assays, and molecular dynamics simulations to investigate the significance of hydrogen bonding interactions that are theorized to stabilize a static active site water network. The active site residue Asn-85, which hydrogen bonds with the diketopiperazine ring of cYY and contributes to a contiguous 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 resulted in inactivation of the enzyme. Moreover, the N85S mutation induced 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 confirmed that disruption of the active site water network also significantly altered the recognition of cYY and the structure of the active site. These data were consistent with dynamics simulations of N85S and N85Q that demonstrated that a compromised water network is responsible for remodeling of the active site B-helix and a repositioning of cYY toward the heme. An Additional mutation that was theorized to alter water interactions within the active site (F168Y) was also found to inactivate CYP121. We conclude that these findings implicate a slowly exchanging water network as a critical factor in CYP121 function and a likely contributor to the unusual rigidity of its structure. This work was supported by SUNY Start-up funds.