Unusual Diheme Conformation of the Heme-Degrading Protein from Mycobacterium tuberculosis

Abstract

Heme degradation plays a pivotal role in the availability of the essential nutrient, iron, in pathogenic bacteria. A previously unannotated protein from Mycobacterium tuberculosis, Rv3592, which shares homology to heme-degrading enzymes, has been identified. Biochemical analyses confirm that Rv3592, which we have termed MhuD (mycobacterial heme utilization, degrader), is able to bind and degrade heme. Interestingly, contrary to previously reported stoichiometry for the Staphylococcus aureus heme degraders, IsdG and IsdI, MhuD has the ability to bind heme in a 1:2 protein to heme ratio, although the MhuD-diheme complex is inactive. Furthermore, the 1.75 Å crystal structure of the MhuD-diheme complex reveals two stacked hemes forming extensive contacts with residues in the active site. In particular, the solvent-exposed heme is axially liganded by His75 and is stacked planar upon the solvent-protected heme. The solvent-protected heme is coordinated by a chloride ion which is, in turn, stabilized by Asn7. Structural comparison between MhuD-diheme and inactive IsdG and IsdI bound to only one highly distorted metalloporphyrin ring reveal that several residues located in α-helix 2 and the subsequent loop appear to be responsible for heme stoichiometric differences and suggest open and closed conformations for substrate entry and product exit.

Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation

“…MhuD can bind up to two molecules of heme in its catalytic center while the active form is the 1:1 complex (Fig. S1 A and B) (15). The monoheme-MhuD complex was selectively prepared by incubating the purified protein in 20 mM Tris·HCl, pH 8.0 containing 10 mM NaCl with 1.0 molar equivalent of hemin at 4°C for 12 h. The crude complex was loaded on an anion exchange column (DE52, Whatman) equilibrated with 0.1 M potassium phosphate, pH 7.0 and the monoheme complex was eluted with the same buffer containing 350 mM NaCl.…”

“…1C and Fig. S1) (15,16). The ruffling is expected to drastically modulate the hemedependent O 2 activation to initiate this unique mechanism (14).…”

Overcoming the Heme Paradox: Heme Toxicity and Tolerance in Bacterial Pathogens

“…IsdG family heme oxygenases have been characterized for S. aureus, B. anthracis, B. japonicum, Mycobacterium tuberculosis, and Brucella melitensis and are predicted to be encoded in the Alphaproteobacteria, Streptomyces, Deinococcus-Thermus, and Chloroflexi groups (17,89,101,102). S. aureus encodes two IsdG family heme oxygenases (102).…”

Characterization of a Mycobacterium tuberculosis Nanocompartment and Its Potential Cargo Proteins

“…Mycobacteria possess a heme uptake pathway (20, 41) along with a cytosolic heme-degrading enzyme, MhuD, which has been shown to degrade heme by cleavage of the tetrapyrrole ring to release iron and mycobilins as products (42,43). To test for Mt-DyP deferrochelatase activity, we utilized a mycobactindeficient M. tuberculosis strain, Mtb⌬mbtB, which has an interrupted mycobactin biosynthetic pathway to prevent siderophore-mediated iron acquisition (20,41) and cannot utilize Fe(III) in the absence of exogenous siderophore (mycobactin).…”