The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands “moonlight” as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

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The thermophilic species, Thermococcus kodakarensis KOD1, a model microorganism for studying hyperthermophiles, has adapted to optimal growth under conditions of high temperature and salinity. However, the environmental conditions for the strain are not always stable, and this strain might face different stresses. In the present study, we compared the proteome response of T. kodakarensis to heat, oxidative, and salt stresses using two-dimensional electrophoresis, and protein spots were identified through MALDI-TOF/MS. Fifty-nine, forty-two, and twenty-nine spots were induced under heat, oxidative, and salt stresses, respectively. Among the up-regulated proteins, four proteins (a hypothetical protein, pyridoxal biosynthesis lyase, peroxiredoxin, and protein disulphide oxidoreductase) were associated with all three stresses. Gene ontology analysis showed that these proteins were primarily involved metabolic and cellular processes. The KEGG pathway analysis suggested that the main metabolic pathways involving these enzymes were related to carbohydrate metabolism, secondary metabolite synthesis, and amino acid biosynthesis. These data might enhance our understanding of the functions and molecular mechanisms of thermophilic Archaea for survival and adaptation in extreme environments.