Mycofactocin is a putative, peptide derived, cofactor that is associated primarily with the Mycobacterium genera including the pathogen M. tuberculosis. The pathway consists of the three genes mftA, mftB, and mftC that encode for the peptide substrate, peptide chaperone, and a radical S-adenosylmethionine protein (RS), respectively. Here, we show that the MftB acts as a peptide chaperone, binding MftA with a submicromolar K D (~100 nM) and MftC with a low micromolar K D (~2 lM). Moreover, we demonstrate that MftC is a radical S-adenosylmethionine (SAM) enzyme. Finally, we show that MftC catalyzes the oxidative decarboxylation of the peptide MftA.
Hemoglobin degradation/hemozoin formation, essential steps in the Plasmodium life cycle, are targets of existing antimalarials. The pathway still offers vast possibilities to be explored for new antimalarial discoveries. Here, we characterize heme detoxification protein, PfHDP, a major protein involved in hemozoin formation, as a novel drug target. Using in silico and biochemical approaches, we identified two heme binding sites and a hemoglobin binding site in PfHDP. Treatment of Plasmodium falciparum 3D7 parasites with peptide corresponding to the hemoglobin binding domain in PfHDP resulted in food vacuole abnormalities similar to that seen with a cysteine protease inhibitor, E-64 (I-1). Screening of compounds that bound the modeled PfHDP structure in the heme/hemoglobin-binding pockets from Maybridge Screening Collection identified a compound, ML-2, that inhibited parasite growth in a dose-dependent manner, thus paving the way for testing its potential as a new drug candidate. These results provide functional insights into the role of PfHDP in Hz formation and further suggest that PfHDP could be an important drug target to combat malaria.
Polymeric nanohybrid P22 virus capsids were used as templates for high density Gd3+ loading to explore magnetic field-dependent (0.5–7.0 T) proton relaxivity. The field-dependence of relaxivity by the spatially constrained Gd3+ in the capsids was similar when either the loading of the capsids or the concentration of capsids was varied. The ionic longitudinal relaxivity, r1, decreased from 25–32 mM−1 s−1 at 0.5 T to 6–10 mM−1 s−1 at 7 T. The ionic transverse relaxivity, r2, increased from 28–37 mM−1 s−1 at 0.5 T to 39–50 mM−1 s−1 at 7 T. The r2/r1 ratio increased linearly with increasing magnetic field from about 1 at 0.5 T, which is typical of T1 contrast agents, to 5–8 at 7 T, which is approaching the ratios for T2 contrast agents. Increases in electron paramagnetic resonance line widths at 80 and 150 K and higher microwave powers required for signal saturation indicate enhanced Gd3+ electron spin relaxation rates for the Gd3+-loaded capsids than for low concentration Gd3+. The largest r2/r1 at 7 T was for the highest cage loading, which suggests that Gd3+–Gd3+ interactions within the capsid enhance r2 more than r1.
Nature utilizes [FeFe]-hydrogenase
enzymes to catalyze the interconversion
between H2 and protons and electrons. Catalysis occurs
at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated
2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis
of this unique metallocofactor is accomplished by three maturase enzymes
denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize
the nonprotein ligands of the H-cluster. These enzymes interact with
HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe
subcluster assembly. Prior characterization of HydF demonstrated the
protein exists in both dimeric and tetrameric states and coordinates
both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters
[Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick,
J. B. (2016) Biochemistry 55, 3514–3527].
Herein, electron paramagnetic resonance (EPR) is utilized to characterize
the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to
HydF. Examination of spin relaxation times using pulsed EPR in HydF
samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types
either are bound to widely separated sites on HydF or are not simultaneously
bound to a single HydF species. Gel filtration chromatographic analyses
of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric
form of the protein. Lastly, we examined the 2Fe subcluster-loaded
form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase
activation. Together, the results indicate a specific role for the
HydF dimer in the H-cluster biosynthesis pathway.
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