Staphylococcus aureus IsdG and IsdI, Heme-degrading Enzymes with Structural Similarity to Monooxygenases

Abstract

Heme-degrading enzymes are involved in human diseases ranging from stroke, cancer, and multiple sclerosis to infectious diseases such as malaria, diphtheria, and meningitis. All mammalian and microbial enzymes identified to date are members of the heme oxygenase superfamily and assume similar monomeric structures with an all ␣-helical fold. Here we describe the crystal structures of IsdG and IsdI, two heme-degrading enzymes from Staphylococcus aureus. The structures of both enzymes resemble the ferredoxin-like fold and form a ␤-barrel at the dimer interface. Two large pockets found on the outside of the barrel contain the putative active sites. Sequence homologs of IsdG and IsdI were identified in multiple Gram-positive pathogens. Substitution of conserved IsdG amino acid residues either reduced or abolished heme degradation, suggesting a common catalytic mechanism. This mechanism of IsdG-mediated heme degradation may be similar to that of the structurally related monooxygenases, enzymes involved in the synthesis of antibiotics in Streptomyces. Our results imply the evolutionary adaptation of microbial enzymes to unique environments.Staphylococcus aureus acquires iron, an essential nutrient required for infection, by binding host heme-carrying proteins and extracting and transporting heme across the bacterial cell wall and plasma membrane envelope (1-3). Once inside the staphylococcal cytoplasm, heme is either incorporated into bacterial heme proteins or degraded to release iron for subsequent incorporation into polypeptides and cofactors (4 -6). One heme acquisition system, encoded by the isd (iron-regulated surface determinants) gene cluster, encodes the heme-degrading enzyme IsdG (1, 5). IsdI, a homolog of IsdG, is encoded elsewhere on the staphylococcal chromosome (5). IsdG and IsdI show no significant sequence similarity to known heme oxygenases (7-10) and do not contain the conserved N-terminal histidine or the GXXXG motif characteristic for these enzymes. Nonetheless, purified IsdG and IsdI cleave heme tetrapyrrole in the presence of suitable electron donors (2, 5). This activity can functionally substitute for the classical heme oxygenase activity and permits growth of Corynebacterium ulcerans (5) lacking the HmuO heme oxygenase on media with hemin as a sole source of iron (8). Here we address the question of whether IsdG and IsdI have structures and catalytic mechanisms similar to those of members of the heme oxygenase superfamily (11), enzymes degrading heme in the cytoplasm of eukaryotic cells and in some bacterial species such as Corynebacterium diphtheriae, Pseudomonas aeruginosa, and Neisseria spp (9). EXPERIMENTAL PROCEDURESPreparation of Proteins for Crystallization-The cells were grown at 37°C in Luria-Bertani broth in the presence of 100 g/ml ampicillin and 30 g/ml kanamycin, respectively. Expression of His-tagged fusion proteins in Escherichia coli strain BL21(DE3) (12) was induced with 1 mM D-isopropyl-␤-thiogalactoside when the optical density at 600 nm reached ϳ0.6 and incubated at 20°C ove...

Bacillus subtilis Fur represses one of two paralogous haem-degrading monooxygenases

“…HmoA and other members of this subfamily (Fig. 1b) contain a basic amino acid (R or K) in place of the N7 in IsdG (Wu et al, 2005). Importantly, members of the HmoA subfamily are found in the human pathogens B. anthracis and Bacillus cereus, where they may play a role in iron acquisition during infection.…”

“…Not surprisingly, the HmoA H76 (equivalent to IsdG H77) is also predicted to be spatially conserved, consistent with its reported role in the binding of the ferric ion of the haem group. However, the essential IsdG N7 residue (Wu et al, 2005), is positionally substituted with R6, as predicted from the sequence alignment (Fig. 2b).…”

“…2b). Since N7 is predicted to interact directly with the haem iron in the active site and is essential for enzymic function (Wu et al, 2005), this substitution is surprising. The replacement of N7(IsdG) with R6(HmoA) suggests that the details of substrate binding or catalysis may differ between the IsdG/HmoB type enzymes and HmoA.…”

“…The haem binding site in IsdG was identified on the basis of mutational analysis as involving residues N7, W67 and H77 (Wu et al, 2005). This critical NWH triad is conserved in HmoB but, surprisingly, only partially conserved in HmoA: the residue corresponding to asparagine 7 (N7) is substituted with arginine (Fig.…”

“…All four mutant proteins bound haem with approximately 1 : 1 stoichiometry, as noted for wild-type HmoA and HmoB. Before, an N7A mutant of S. aureus IsdG has been shown to still bind haem but to have lost all capacity for haem degradation (Wu et al, 2005). In contrast, both the Haemin was added to both the sample and reference cuvettes and the differences in absorbance were recorded.…”