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.

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

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.

Crystal Structures of Drug-Metabolizing CYPs.

The complex enzyme kinetics displayed by drug-metabolizing cytochrome P450 enzymes (CYPs) (see Chapter 9 ) can, in part, be explained by an examination of their crystallographic protein structures. Fortunately, despite low sequence similarity between different families of drug-metabolizing CYPs, there exists a high degree of structural homology within the superfamily. This similarity in the protein fold allows for a direct comparison of the structural features of CYPs that contribute toward differences in substrate binding, heterotropic and homotropic cooperativity, and genetic variability in drug metabolism. In this chapter, we first provide an overview of the nomenclature and the role of structural features that are common in all CYPs. We then apply these definitions to understand the different substrate specificities and functions in the CYP3A, CYP2C, and CYP2D families of enzymes.

Surface hydrophobics mediate functional dimerization of CYP121A1 of Mycobacterium tuberculosis.

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. The Mtb genome encodes a disproportionate number of twenty cytochrome P450 enzymes, of which the essential enzyme cytochrome P450 121A1 (CYP121A1) remains a target of drug design efforts. CYP121A1 mediates a phenol coupling reaction of the tyrosine dipeptide cyclo-L-Tyr-L-Tyr (cYY). In this work, a structure and function investigation of dimerization was performed as an overlooked feature of CYP121A1 function. This investigation showed that CYP121A1 dimers form via intermolecular contacts on the distal surface and are mediated by a network of solvent-exposed hydrophobic residues. Disruption of CYP121A1 dimers by site-directed mutagenesis leads to a partial loss of specificity for cYY, resulting in an approximate 75% decrease in catalysis. 19F labeling and nuclear magnetic resonance of the enzyme FG-loop was also combined with protein docking to develop a working model of a functional CYP121A1 dimer. The results obtained suggest that participation of a homodimer interface in substrate selectivity represents a novel paradigm of substrate binding in CYPs, while also providing important mechanistic insight regarding a relevant drug target in the development of novel anti-tuberculosis agents.

Translational Regulation Promotes Oxidative Stress Resistance in the Human Fungal Pathogen Cryptococcus neoformans.

Cryptococcus neoformans is one of the few environmental fungi that can survive within a mammalian host and cause disease. Although many of the factors responsible for establishing virulence have been recognized, how they are expressed in response to certain host-derived cellular stresses is rarely addressed. Here, we characterize the temporal translational response of C. neoformans to oxidative stress. We find that translation is largely inhibited through the phosphorylation of the critical initiation factor eIF2α (α subunit of eukaryotic initiation factor 2) by a sole kinase. Preventing eIF2α-mediated translational suppression resulted in growth sensitivity to hydrogen peroxide (H2O2). Our work suggests that translational repression in response to H2O2 partly facilitates oxidative stress adaptation by accelerating the decay of abundant non-stress-related transcripts while facilitating the proper expression levels of select oxidative stress response factors. Our results illustrate translational suppression as a critical determinant of select mRNA decay, gene expression, and subsequent survival in response to oxidative stress.IMPORTANCE Fungal survival in a mammalian host requires the coordinated expression and downregulation of a large cohort of genes in response to cellular stresses. Initial infection with C. neoformans occurs in the lungs, where it interacts with host macrophages. Surviving macrophage-derived cellular stresses, such as the production of reactive oxygen and nitrogen species, is believed to promote dissemination into the central nervous system. Therefore, investigating how an oxidative stress-resistant phenotype is brought about in C. neoformans not only furthers our understanding of fungal pathogenesis but also unveils mechanisms of stress-induced gene reprogramming. We discovered that H2O2-derived oxidative stress resulted in severe translational suppression and that this suppression was necessary for the accelerated decay and expression of tested transcripts.