The human pathogen Mycobacterium tuberculosis (Mtb) encodes 20 cytochrome P450 (P450) enzymes. Gene essentiality for viability or host infection was demonstrated for Mtb P450s CYP128, CYP121 and CYP125. Structure/function studies on Mtb P450s revealed key roles contributing to bacterial virulence and persistence in the host. Various azole-class drugs bind with high affinity to the Mtb P450 heme and are potent Mtb antibiotics. This paper reviews the current understanding of the biochemistry of Mtb P450s, their interactions with azoles and their potential as novel Mtb drug targets. Mtb multidrug resistance is widespread and novel therapeutics are desperately needed. Simultaneous drug targeting of several Mtb P450s crucial to bacterial viability/persistence could offer a new route to effective antibiotics and minimize the development of drug resistance.
The cytochrome P450/P450 reductase
fusion enzyme CYP505A30 from
the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia
coli and purified using affinity, ion exchange, and
size exclusion chromatography. CYP505A30 binds straight chain fatty
acids (from ∼C10 to C20), with highest affinity for tridecanoic
acid (KD = 2.7 μM). Reduced nicotinamide
adenine dinucleotide phosphate is the preferred reductant for CYP505A30
(KM = 3.1 μM compared to 330 μM
for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed
cysteine thiolate coordination of heme iron in CYP505A30 and its heme
domain. Redox potentiometry revealed an unusually positive midpoint
potential for reduction of the flavin adenine dinucleotide and flavin
mononucleotide cofactors (E0′ ∼
−118 mV), and a large increase in the CYP505A30 heme domain
FeIII/FeII redox couple (ca. 230 mV) on binding
arachidonic acid substrate. This switch brings the ferric heme iron
potential into the same range as that of the reductase flavins. Multiangle
laser light scattering analysis revealed CYP505A30’s ability
to dimerize, whereas the heme domain is monomeric. These data suggest
CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions
between CYP505A30 heme domains are not required for dimer formation.
CYP505A30 catalyzed hydroxylation of straight chain fatty acids at
the ω-1 to ω-3 positions, with a strong preference for
ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic
and tetradecanoic acids (88 vs 2% products and 63 vs 9% products,
respectively). CYP505A30 has important structural and catalytic similarities
to P450 BM3 but distinct regioselectivity of lipid substrate oxidation
with potential biotechnological applications.
The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.
Flavocytochrome P450 BM3 is a natural fusion protein constructed of cytochrome P450 and NADPH-cytochrome P450 reductase domains. P450 BM3 binds and oxidizes several mid- to long-chain fatty acids, typically hydroxylating these lipids at the ω-1, ω-2 and ω-3 positions. However, protein engineering has led to variants of this enzyme that are able to bind and oxidize diverse compounds, including steroids, terpenes and various human drugs. The wild-type P450 BM3 enzyme binds inefficiently to many azole antifungal drugs. However, we show that the BM3 A82F/F87V double mutant (DM) variant binds substantially tighter to numerous azole drugs than does the wild-type BM3, and that their binding occurs with more extensive heme spectral shifts indicative of complete binding of several azoles to the BM3 DM heme iron. We report here the first crystal structures of P450 BM3 bound to azole antifungal drugs – with the BM3 DM heme domain bound to the imidazole drugs clotrimazole and tioconazole, and to the triazole drugs fluconazole and voriconazole. This is the first report of any protein structure bound to the azole drug tioconazole, as well as the first example of voriconazole heme iron ligation through a pyrimidine nitrogen from its 5-fluoropyrimidine ring.
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