Abstract
The Rv2633c gene in Mycobacterium tuberculosis is rapidly up-regulated after macrophage infection, suggesting that Rv2633c is involved in M. tuberculosis pathogenesis. However, the activity and role of the Rv2633c protein in host colonization is unknown. Here, we analyzed the Rv2633c protein sequence, which revealed the presence of an HHE cation-binding domain common in hemerythrin-like proteins. Phylogenetic analysis indicated that Rv2633c is a member of a distinct subset of hemerythrin-like proteins exclusive to mycobacteria. The Rv2633c sequence was significantly similar to protein sequences from other pathogenic strains within that subset, suggesting that these proteins are involved in mycobacteria virulence. We expressed and purified the Rv2633c protein in Escherichia coli and found that it contains two iron atoms, but does not behave like a hemerythrin. It migrated as a dimeric protein during size-exclusion chromatography. It was not possible to reduce the protein or observe any evidence for its interaction with O2 However, Rv2633c did exhibit catalase activity with a kcat of 1475 s-1 and Km of 10.1 ± 1.7 mm Cyanide and azide inhibited the catalase activity with Ki values of 3.8 μm and 37.7 μm, respectively. Rv2633c's activity was consistent with a role in defenses against oxidative stress generated during host immune responses after M. tuberculosis infection of macrophages. We note that Rv2633c is the first example of a non-heme di-iron catalase, and conclude that it is a member of a subset of hemerythrin-like proteins exclusive to mycobacteria, with likely roles in protection against host defenses.
Highlights
The Rv2633c gene in Mycobacterium tuberculosis is rapidly up-regulated after macrophage infection, suggesting that Rv2633c is involved in M. tuberculosis pathogenesis
We note that Rv2633c is the first example of a non-heme di-iron catalase, and conclude that it is a member of a subset of hemerythrin-like proteins exclusive to mycobacteria, with likely roles in protection against host defenses
Analysis of the sequence of the protein encoded by the Rv2633c gene, which is presented in this paper, reveals the presence of an HHE cation-binding domain that is common in hemerythrins and hemerythrin-like proteins
Summary
Inspection of the primary sequence of Rv2633c revealed the presence of an HHE cation-binding domain that is common in hemerythrins (Fig. 1). No other species of bacteria have a gene product with statistically significant homology across the entire protein sequence This analysis strongly suggests that the gene product of Rv2633c is involved in the unique ability of opportunistic and authentic pathogens of this clade. Rv2633c showed no reactivity toward O2, as judged by the lack of change of the absorbance spectrum after removal of the excess reductant and exposure to air These results indicate that the protein environment of the metal site strongly stabilizes the diferric state of the protein. Catalase activity of Rv2633c was suspected as formation of bubbles and foam was observed on addition of H2O2 to solutions of the protein (Fig. 5). No peroxidase activity was observed using either ascorbate, pyrogallol or o-dianisidine as an electron donor at concentrations up to 100 mM
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