The structural characterization of bacterioferritin, BfrA, from Mycobacterium tuberculosis

Abstract

Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. Like most bacterial pathogens, iron acquisition, regulation, and storage are critical for its survival. Due to the poor solubility of iron under physiological conditions, both eukaryotes and prokaryotes possess ferritins, large protein complexes that store iron and keep it bioavailable. Mtb encodes for two ferritin homologs: a hemecontaining bacterioferritin (Mtb-BfrA) and a non-heme eukaryotic-like ferritin (Mtb-BfrB). A conserved feature of bacterioferritins is the presence of a heme group at the interface between two subunits of each dimer that is related by a non-crystallographic two-fold axis. The structure of a selenomethionine derivative of Mtb-BfrA was previously reported (PDB ID: 2WTL); however, a proposed heme degradation product was modeled into the heme-binding site, as electron density for intact heme was not observed. Here, the purification and structure determination of recombinant Mtb-BfrA is reported. As-isolated Mtb-BfrA from Escherichia coli is not fully heme loaded. However, the absorption spectrum features suggest binding of intact heme. In an attempt to fully complement Mtb-BfrA with heme, two different methodologies are described. Electronic spectroscopy and structure determination were used to confirm varying amounts of intact bis-methionine coordinated heme to Mtb-BfrA. We also report that increased heme incorporation only slightly increases Mtb-BfrA ferroxidase activity. Finally, the cognate partner of Mtb-BfrA is proposed to be a putative encoded gene which is located approximately 300 bps upstream of Mycobacterium tuberculosis bfrA, homologous to the cognate partner of Pseudomonas aeruginosa bacterioferritin, a 7 kDa ferrodoxin Bfd.

Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in Mycobacterium tuberculosis

“…Besides, BfrA is a heme binding protein and heme is often reported to be associated with the iron release process from the bacterioferritins [27, 28]. Met52 is a conserved residue which is known to be involved in heme binding in bacterioferritins [27, 28, 29]. Hence, we also generated a site directed mutant of BfrA wherein Met at position 52 was replaced with leucine (BfrAM52L).…”

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

“…Mt-DyP has a 50-fold lower k cat value than a DyP from a white rot fungus Phanerochaete chrysosporium (37); however, it has been noted that M. tuberculosis enzymes may have slower rates of reactions compared with their homologs (38,39). Second, utilizing ABTS as the reducing substrate, holo-Mt-DyP produced an ABTS radical cation in the presence of H 2 O 2 indicative of an active peroxidase, whereas apo-MtDyP displayed background activity (Fig.…”

“…6E). The other M. tuberculosis ferritin homolog, Mt-BfrA, also selfassembles into a 24-subunit nanocage (38,39); however, MtBfrA does not possess a C-terminal extension. When Mt-BfrA was coexpressed with Mt-Enc followed by affinity chromatography purification, it did not co-elute with Mt-Enc (Fig.…”

Agrobacterium tumefaciens ferritins play an important role in full virulence through regulating iron homeostasis and oxidative stress survival

“…Therefore, we first conducted a bioinformatics analysis on the putative bfr gene and its product Bfr protein. In the genomes of many bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and so on, the bfr gene is often adjacent to a bfd gene, which encodes a bacterioferritin-associated ferredoxin (Bfd) (Yao et al, 2012;McMath et al, 2013;Eshelman et al, 2017) (Figure 1a). The Bfd protein is suggested to be involved in the delivery of electrons from NADP-ferredoxin reductase to haem in the process of iron release from Bfr (Yao et al, 2012;Wang et al, 2015;Eshelman et al, 2017;Punchi Hewage et al, 2019).…”