Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biological function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivatives of PA-824 were used, the amount of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compounds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system.

Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm. Less relevant is the selective incorporation of (15)N. Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications.

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The ligand binding and catalytic properties of heavy metal ions have led to the evolution of metal ion-specific pathways for control of their intracellular trafficking and/or elimination. Small MW proteins/domains containing a GMTCXXC metal binding motif in a betaalphabetabetaalphabeta fold are common among proteins controlling the mobility of soft metal ions such as Cu(1+), Zn(2+), and Hg(2+), and the functions of several have been established. In bacterial mercuric ion reductases (MerA), which catalyze reduction of Hg(2+) to Hg(0) as a means of detoxification, one or two repeats of sequences with this fold are highly conserved as N-terminal domains (NmerA) of uncertain function. To simplify functional analysis of NmerA, we cloned and expressed the domain and catalytic core of Tn501 MerA as separate proteins. In this paper, we show Tn501 NmerA to be a stable, soluble protein that binds 1 Hg(2+)/domain and delivers it to the catalytic core at kinetically competent rates. Comparison of steady-state data for full-length versus catalytic core MerA using Hg(glutathione)(2) or Hg(thioredoxin) as substrate demonstrates that the NmerA domain does participate in acquisition and delivery of Hg(2+) to the catalytic core during the reduction catalyzed by full-length MerA, particularly when Hg(2+) is bound to a protein. Finally, comparison of growth curves for glutathione-depleted Escherichia coli expressing either catalytic core, full-length, or a combination of core plus NmerA shows an increased protection of cells against Hg(2+) in the media when NmerA is present, providing the first evidence of a functional role for this highly conserved domain.