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• W1971624564 abstract "Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a Km of 15 ± 4 μm, a kcat of 5.2 ± 0.7 min−1, and a kcat/Km of 5.8 × 103 m−1 s−1. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.Background: Staphylococcus aureus utilizes heme as an iron source during an infection.Results: An oxidoreductase, IruO, can supply electrons to IsdI and IsdG for heme degradation and iron extraction.Conclusion: IruO is likely the in vivo reductant for heme degradation to the staphylobilins.Significance: Heme degradation is a potential target for anti-S. aureus therapeutics. Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a Km of 15 ± 4 μm, a kcat of 5.2 ± 0.7 min−1, and a kcat/Km of 5.8 × 103 m−1 s−1. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus. Background: Staphylococcus aureus utilizes heme as an iron source during an infection. Results: An oxidoreductase, IruO, can supply electrons to IsdI and IsdG for heme degradation and iron extraction. Conclusion: IruO is likely the in vivo reductant for heme degradation to the staphylobilins. Significance: Heme degradation is a potential target for anti-S. aureus therapeutics. Staphylococcus aureus is a Gram-positive pathogen that causes a diverse range of infections from skin and soft tissue infections to necrotizing pneumonia and fasciitis using many virulence factors (1.Gordon R.J. Lowy F.D. Pathogenesis of methicillin-resistant Staphylococcus aureus infection.Clin. Infect. Dis. 2008; 46: S350-S359Crossref PubMed Scopus (657) Google Scholar, 2.Lowy F.D. Staphylococcus aureus infections.N. Engl. J. Med. 1998; 339: 520-532Crossref PubMed Scopus (4597) Google Scholar). S. aureus can be acquired either in the community or nosocomially, and many pathogenic strains are multidrug resistant, leaving a limited number of treatment options available (3.Thurlow L.R. Joshi G.S. Richardson A.R. Virulence strategies of the dominant USA300 lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA).FEMS Immunol. Med. Microbiol. 2012; 65: 5-22Crossref PubMed Scopus (103) Google Scholar). Furthermore, drug-resistant strains have spread throughout the world (4.Nimmo G.R. USA300 abroad. Global spread of a virulent strain of community-associated methicillin-resistant Staphylococcus aureus.Clin. Microbiol. Infect. 2012; 18: 725-734Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), leading to a need for the characterization of S. aureus pathways required for infectivity as a foundation to new human therapies. Like almost all bacteria, S. aureus requires a source of iron for bacterial metabolism and growth. Within mammalian hosts, the concentration of iron freely available to S. aureus is negligible as iron is found either intracellularly as protein cofactors or complexed by host proteins such as transferrin and lactoferrin (5.Bullen J.J. Rogers H.J. Spalding P.B. Ward C.G. Iron and infection. The heart of the matter.FEMS Immunol. Med. Microbiol. 2005; 43: 325-330Crossref PubMed Scopus (204) Google Scholar). This iron sequestration is a form of nutritional immunity that protects mammals from infection (6.Weinberg E.D. Infection and iron metabolism.Am. J. Clin. Nutr. 1977; 30: 1485-1490Crossref PubMed Scopus (41) Google Scholar). Consequently, S. aureus has evolved multiple strategies for iron acquisition (7.Hammer N.D. Skaar E.P. Molecular mechanisms of Staphylococcus aureus iron acquisition.Annu. Rev. Microbiol. 2011; 65: 129-147Crossref PubMed Scopus (232) Google Scholar). S. aureus produces two siderophores, staphyloferrin A (8.Beasley F.C. Vinés E.D. Grigg J.C. Zheng Q. Liu S. Lajoie G.A. Murphy M.E. Heinrichs D.E. Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus.Mol. Microbiol. 2009; 72: 947-963Crossref PubMed Scopus (105) Google Scholar, 9.Cotton J.L. Tao J. Balibar C.J. Identification and characterization of the Staphylococcus aureus gene cluster coding for Staphyloferrin A.Biochemistry. 2009; 48: 1025-1035Crossref PubMed Scopus (63) Google Scholar) and staphyloferrin B (10.Cheung J. Beasley F.C. Liu S. Lajoie G.A. Heinrichs D.E. Molecular characterization of staphyloferrin B biosynthesis in Staphylococcus aureus.Mol. Microbiol. 2009; 74: 594-608Crossref PubMed Scopus (88) Google Scholar), and has a transport system that can co-opt hydroxamate-type siderophores produced by other bacteria (11.Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus.J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (108) Google Scholar). S. aureus can also obtain heme from host heme-containing proteins hemoglobin and haptoglobin, transport it across the bacterial cell envelope, cleave the porphyrin ring, and release iron for use by the cell with the well characterized iron-regulated surface determinant (Isd) 3The abbreviations used are: Isdiron-regulated surface determinantIruOiron utilization oxidoreductasePNDOpyridine nucleotide-disulfide oxidoreductaseTCEPTris(2-carboxyethyl)phosphineFurferric-uptake regulatorBis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolTrxthioredoxin reductase. system (12.Grigg J.C. Ukpabi G. Gaudin C.F. Murphy M.E. Structural biology of heme binding in the Staphylococcus aureus Isd system.J. Inorg. Biochem. 2010; 104: 341-348Crossref PubMed Scopus (107) Google Scholar). A series of cell wall-anchored proteins (IsdA, IsdB, IsdC, and IsdH) bind host heme-containing proteins, extract heme, and shuttle it to the bacterial membrane (13.Muryoi N. Tiedemann M.T. Pluym M. Cheung J. Heinrichs D.E. Stillman M.J. Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.J. Biol. Chem. 2008; 283: 28125-28136Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 14.Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Passage of heme-iron across the envelope of Staphylococcus aureus.Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 15.Torres V.J. Pishchany G. Humayun M. Schneewind O. Skaar E.P. Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization.J. Bacteriol. 2006; 188: 8421-8429Crossref PubMed Scopus (219) Google Scholar, 16.Vermeiren C.L. Pluym M. Mack J. Heinrichs D.E. Stillman M.J. Characterization of the heme binding properties of Staphylococcus aureus IsdA.Biochemistry. 2006; 45: 12867-12875Crossref PubMed Scopus (62) Google Scholar, 17.Pishchany G. Dickey S.E. Skaar E.P. Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.Infect. Immun. 2009; 77: 2624-2634Crossref PubMed Scopus (57) Google Scholar, 18.Tiedemann M.T. Heinrichs D.E. Stillman M.J. The multi-protein heme shuttle pathway in Staphylococcus aureus. Isd cog-wheel kinetics.J. Am. Chem. Soc. 2012; 134: 16578-16585Crossref PubMed Scopus (32) Google Scholar, 19.Dryla A. Gelbmann D. von Gabain A. Nagy E. Identification of a novel iron regulated staphylococcal surface protein with haptoglobin-haemoglobin binding activity.Mol. Microbiol. 2003; 49: 37-53Crossref PubMed Scopus (126) Google Scholar). There an ABC transporter consisting of IsdE, IsdF, and possibly IsdD moves heme across the membrane and into the cytoplasm (14.Mazmanian S.K. Skaar E.P. Gaspar A.H. Humayun M. Gornicki P. Jelenska J. Joachmiak A. Missiakas D.M. Schneewind O. Passage of heme-iron across the envelope of Staphylococcus aureus.Science. 2003; 299: 906-909Crossref PubMed Scopus (465) Google Scholar, 20.Grigg J.C. Vermeiren C.L. Heinrichs D.E. Murphy M.E. Heme coordination by Staphylococcus aureus IsdE.J. Biol. Chem. 2007; 282: 28815-28822Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21.Pluym M. Vermeiren C.L. Mack J. Heinrichs D.E. Stillman M.J. Heme binding properties of Staphylococcus aureus IsdE.Biochemistry. 2007; 46: 12777-12787Crossref PubMed Scopus (35) Google Scholar). Once in the cytoplasm, two paralogous (64% amino acid sequence identity) but differentially regulated proteins (IsdG and IsdI) have the ability to cleave the porphyrin ring of heme and release iron (22.Skaar E.P. Gaspar A.H. Schneewind O. IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus.J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 23.Wu R. Skaar E.P. Zhang R. Joachimiak G. Gornicki P. Schneewind O. Joachimiak A. Staphylococcus aureus IsdG and IsdI, heme-degrading enzymes with structural similarity to monooxygenases.J. Biol. Chem. 2005; 280: 2840-2846Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 24.Reniere M.L. Skaar E.P. Staphylococcus aureus haem oxygenases are differentially regulated by iron and haem.Mol. Microbiol. 2008; 69: 1304-1315Crossref PubMed Scopus (63) Google Scholar). The Isd pathway is important for the pathogenesis of S. aureus as heme may be the preferred iron source (25.Skaar E.P. Humayun M. Bae T. DeBord K.L. Schneewind O. Iron-source preference of Staphylococcus aureus infections.Science. 2004; 305: 1626-1628Crossref PubMed Scopus (317) Google Scholar), and IsdB and IsdE have both been implicated in systemic infections of mice (17.Pishchany G. Dickey S.E. Skaar E.P. Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.Infect. Immun. 2009; 77: 2624-2634Crossref PubMed Scopus (57) Google Scholar, 26.Mason W.J. Skaar E.P. Assessing the contribution of heme-iron acquisition to Staphylococcus aureus pneumonia using computed tomography.PLoS ONE. 2009; 4: e6668Crossref PubMed Scopus (22) Google Scholar). iron-regulated surface determinant iron utilization oxidoreductase pyridine nucleotide-disulfide oxidoreductase Tris(2-carboxyethyl)phosphine ferric-uptake regulator 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol thioredoxin reductase. In vitro cleavage of the porphyrin ring by IsdG or IsdI requires molecular oxygen and a source of electrons, and ascorbic acid or non-S. aureus reductase proteins have typically been used as the electron donor (22.Skaar E.P. Gaspar A.H. Schneewind O. IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus.J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). IsdG and IsdI cleave the porphyrin ring at either the δ-meso or β-meso carbons, resulting in two different products, 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin, that are known as the staphylobilins. They are similar to but distinct from biliverdin, the product of heme degradation by conventional heme oxygenases such as human heme oxygenase (HO-1), suggesting that the reaction mechanism is different (27.Reniere M.L. Ukpabi G.N. Harry S.R. Stec D.F. Krull R. Wright D.W. Bachmann B.O. Murphy M.E. Skaar E.P. The IsdG-family of haem oxygenases degrades haem to a novel chromophore.Mol. Microbiol. 2010; 75: 1529-1538Crossref PubMed Scopus (107) Google Scholar). Unlike HO-1, which generates CO during heme degradation, IsdG and IsdI generate formaldehyde (28.Matsui T. Nambu S. Ono Y. Goulding C.W. Tsumoto K. Ikeda-Saito M. Heme degradation by Staphylococcus aureus IsdG and IsdI liberates formaldehyde rather than carbon monoxide.Biochemistry. 2013; 52: 3025-3027Crossref PubMed Scopus (69) Google Scholar). Heme bound to IsdG and IsdI is significantly distorted from planarity in a fashion described as ruffling (29.Lee W.C. Reniere M.L. Skaar E.P. Murphy M.E. Ruffling of metalloporphyrins bound to IsdG and IsdI, two heme-degrading enzymes in Staphylococcus aureus.J. Biol. Chem. 2008; 283: 30957-30963Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 30.Takayama S.-I.J. Ukpabi G. Murphy M.E. Mauk A.G. Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI.Proc. Natl. Acad. Sci. 2011; 108: 13071-13076Crossref PubMed Scopus (44) Google Scholar); IsdI amino acid variants with decreased heme ruffling capability have decreased heme degradation rates (31.Ukpabi G. Takayama S.-i.J. Mauk A.G. Murphy M.E. Inactivation of IsdI heme oxidation by an active site substitution that diminishes heme ruffling.J. Biol. Chem. 2012; 287: 34179-88179Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Outstanding questions about heme degradation in S. aureus, include how the reaction differs from other heme degrading enzymes to generate these novel products, what is the intracellular fate of the staphylobilins, and what is the in vivo electron donor for the reaction? Here, we show that a protein encoded by NWMN2274 in S. aureus strain Newman can act as a source of electrons for heme degradation by IsdG and IsdI in the presence of NADPH. In vitro heme degradation in the presence of this protein yields the same products as reactions with ascorbic acid as an electron donor. From the specificity of the reaction, NWMN2274 is proposed to be the biological reductase associated with IsdG or IsdI heme degradation within the cytoplasm of S. aureus, and we have named this protein the iron utilization oxidoreductase, or IruO. All chemicals were obtained from Fisher unless noted below. Full-length NWMN2274 was PCR-amplified from S. aureus strain Newman chromosomal DNA with forward primer (5′-AGC GGC CTG GTG CCG CGC GGC AGC ATG AAA GAT GTT ACA ATC ATT GGT-3′) and reverse primer (5′-GCG GCC GCA AGC TTG TCG ACG GAG TTA CTA GTA TAA ATG TTT ATT TAC AAT-3′) to form a megaprimer PCR product. Underlined sequences represent homology to pET28a. The thermal cycling conditions were 98 °C for 1 min, 30 cycles of 98 °C (10 s), 58 °C (30 s), and 72 °C (30 s), and a final extension at 72 °C for 5 min. Megaprimer extension for increased homology to pET28a was performed with forward extension primer 5′-AGC AGC CAT CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC-3′ and reverse extension primer 5′-TGG TGG TGG TGC TCG AGT GCG GCC GCA AGC TTG TCG ACG GAG TTA-3′. The thermal cycling conditions were 98 °C for 1 min, 25 cycles of 98 °C (10 s), 55 °C (20 s), and 72 °C (15 s), and a final extension at 72 °C for 5 min. Both reactions were performed with Phusion High-Fidelity DNA polymerase (New England Biolabs). Insertion of the NWMN2274 amplicon into pET28a was performed using a previously described whole plasmid PCR technique (32.MacPherson I.S. Rosell F.I. Scofield M. Mauk A.G. Murphy M.E. Directed evolution of copper nitrite reductase to a chromogenic reductant.Protein Eng. Des. Sel. 2010; 23: 137-145Crossref PubMed Scopus (21) Google Scholar). The pET28a-NWMN2274 construct was introduced into Escherichia coli BL21(λDE3). Colonies containing pET28a-NWMN2274 were confirmed by DNA sequencing. A second PNDO-encoding gene, NWMN0732, was similarly cloned using the primers 5′-AGC GGC CTG GTG CCG CGC GGC AGC ATG ACT GAA ATA GAT TTT GA-3′ and 5′-GCG GCC GCA AGC TTG TCG ACG GAG TTA TTA AGC TTG ATC GTT TAA ATG TTC AAT-3′ to generate pET28a-NWMN0732. For protein expression, E. coli BL21(λDE3) with pET28a-NWMN2274 was grown in 2× YT media supplemented with 25 mg/ml kanamycin at 30 °C to an optical density at 600 nm of ∼0.8. Cultures were then induced with 0.3 mm isopropyl β-d-thiogalactopyranoside and incubated for ∼16 h at 25 °C with shaking at 200 rpm. Cell pellets were collected by centrifugation at 4400 × g for 10 min, resuspended in 50 mm Tris (pH 8.0), 100 mm NaCl, 2 mm Tris(2-carboxyethyl)phosphine (TCEP) (Gold Biotechnology), and lysed at 10,000 p.s.i. with an EmulsiFlex-C5 homogenizer (Avestin). The supernatant was isolated after centrifugation at 39,000 × g for 45 min, and His6-NWMN2274 was purified using a 5-ml HisTrap HP column (GE Healthcare) with a linear imidazole gradient (0–500 mm). Protein fractions were dialyzed into 50 mm Tris-HCl (pH 8.0), 100 mm NaCl, and 2 mm TCEP at 4 °C. The His6 tag was removed by thrombin (Hemotologic Technologies) digestion at a 1/500 (w/w) thrombin-to-protein ratio and incubated over 24 h at 4 °C followed by dialysis into 50 mm Tris-HCl (pH 8.0), 2 mm TCEP for 2 h at 4 °C. NWMN2274 was further purified by anion exchange chromatography using a Source 15Q column (GE Healthcare) equilibrated with 50 mm Tris-HCl (pH 8.0), 2 mm TCEP and eluted with a NaCl gradient (0–500 mm). NWMN2274 was dialyzed into 50 mm Tris (pH 8.0), 300 mm KCl, and 2 mm TCEP and concentrated to 20 mg/ml (Fig. 1D). Purified protein was protected from light and stored at 4 °C. NWMN0732 was purified by using the same protocol. IsdI and IsdG were expressed in E. coli BL21 (λDE3) cells from the plasmid pET15b, purified by His-tag affinity chromatography, and digested with the tobacco etch virus protease to remove the His tag as previously described (22.Skaar E.P. Gaspar A.H. Schneewind O. IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus.J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Tobacco etch virus protease was purified as previously described (33.Tropea J.E. Cherry S. Waugh D.S. Expression and purification of soluble His6-tagged TEV protease.Methods Mol. Biol. 2009; 498: 297-307Crossref PubMed Scopus (250) Google Scholar). Electronic spectra from 250 to 800 nm were measured with a Cary 50 Bio UV-visible spectrophotometer. Samples were NWMN2274 (as purified), NWMN2274 after heat denaturation and protein removal, and FAD, FMN, and riboflavin standards (Sigma). All samples were at 30 μm in a buffer of 50 mm Tris-HCl (pH 8.0), 100 mm NaCl. To heat-denature and remove NWMN2274 from the flavin molecule, the protein solution was boiled for 10 min and centrifuged at 21,000 × g for 3 min, and the supernatant was centrifuged through a Nanosep centrifugal device (PALL Life Sciences) with a molecular mass cut-off of 3 kDa as described previously (34.Aliverti A. Curti B. Vanoni M.A. Identifying and quantitating FAD and FMN in simple and in iron-sulfur-containing flavoproteins.Methods Mol. Biol. 1999; 131: 9-23PubMed Google Scholar). Flavin removed from NWMN2274 as well as FAD, FMN, and riboflavin standards (all at 20 μm) were separated using an Infinity 1260 Quaternary high performance liquid chromatography (HPLC) system (Agilent) equipped with an Aqua 5-mm C18 column (Phenomenex). A previously established procedure was utilized (34.Aliverti A. Curti B. Vanoni M.A. Identifying and quantitating FAD and FMN in simple and in iron-sulfur-containing flavoproteins.Methods Mol. Biol. 1999; 131: 9-23PubMed Google Scholar) with modifications. A flow rate of 1 ml/min and a column temperature of 20 °C were maintained during the entire analysis. The column was equilibrated with 85% solvent A (10 mm ammonium acetate (pH 6.5)) and 15% solvent B (methanol) at sample injection. After a 5-min post-injection period, a linear gradient was developed over 20 min to 75% solvent B followed by a second linear gradient over 5 min to 100% solvent B. Flavins were detected by absorption at 264 nm using an Infinity 1260 multiple wavelength detector (Agilent). In vitro heme degradation assays were conducted as previously described (29.Lee W.C. Reniere M.L. Skaar E.P. Murphy M.E. Ruffling of metalloporphyrins bound to IsdG and IsdI, two heme-degrading enzymes in Staphylococcus aureus.J. Biol. Chem. 2008; 283: 30957-30963Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). 10 mm stock solutions of porcine hemin (Sigma) in 0.1 m NaOH were prepared and kept at −20 °C. 10 μm IsdI and 10 μm porcine hemin (Sigma) were mixed in buffer containing 50 mm Tris-HCl (pH 7.5) and 150 mm NaCl and incubated at 4 °C for 1 h. Spectra from 300 to 800 nm were measured with a Cary 50 Bio UV-visible spectrophotometer. As noted below, some reactions were also supplemented with 200 μm NADPH (EMD Biosciences), 200 μm NADH (Roche), 5 μm bovine liver catalase (Sigma), or 4 units/ml bovine erythrocyte superoxide dismutase (Sigma). To initiate degradation reactions, 1 mm ascorbic acid, 1 μm NWMN2274, or NWMN0732, 20 μm to 2 mm H2O2, or 5 units/ml Aspergillus niger glucose oxidase (Sigma), and 1 mm glucose (Sigma) were added to cuvettes. Spectra were recorded either every minute or every 10 min for up to 90 min depending on the rate at which the reaction progressed. For kinetic analysis, 600 μm NADPH and 0.1 μm NWMN2274 were added to reactions containing 1–25 μm IsdG-heme or IsdI-heme, and the decrease in the Soret peak at 412 nm was monitored every 0.1 s for 180 s. The concentration of IsdI-heme was determined from Soret absorbance measurements for 30 s starting at 10 s after the addition of the reductase using the reported extinction coefficients for IsdG-heme (131 mm−1 cm−1) and IsdI-heme (126 mm−1 cm−1) (22.Skaar E.P. Gaspar A.H. Schneewind O. IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus.J. Biol. Chem. 2004; 279: 436-443Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Slopes determined by linear regressions of these data were taken as initial reaction rates. Initial steady-state rates were plotted against the concentration of IsdG-heme or IsdI-heme, and a non-linear regression of the data were calculated to fit Michaelis-Menten kinetics. Linear and non-linear regressions were calculated using GraphPad Prism 6. For these experiments, 1-ml reactions containing 100 μm IsdI-heme were prepared, and 2 mm ascorbic acid, 2 mm H2O2, 2 units/ml glucose oxidase, and 5 mm glucose or 1 mm NADPH and 5 μm NWMN2274 or NWMN0732 were added to initiate reactions. Reactions were monitored, and once complete, heme degradation products were purified as previously described (27.Reniere M.L. Ukpabi G.N. Harry S.R. Stec D.F. Krull R. Wright D.W. Bachmann B.O. Murphy M.E. Skaar E.P. The IsdG-family of haem oxygenases degrades haem to a novel chromophore.Mol. Microbiol. 2010; 75: 1529-1538Crossref PubMed Scopus (107) Google Scholar) with one important modification. Purification includes sample filtration through a Nanosep centrifugal device. The presence of the larger NWMN2274 protein blocked the pores of spin columns with a molecular mass cutoff of 3 kDa and prevented samples from passing easily through the column. Pore size was increased to a cutoff of 10 kDa, which improved product purification significantly, but lower yields were typically obtained from the NWMN2274/NADPH reactions than from the ascorbic acid reactions. HPLC separation of the degradation products was completed as previously described (27.Reniere M.L. Ukpabi G.N. Harry S.R. Stec D.F. Krull R. Wright D.W. Bachmann B.O. Murphy M.E. Skaar E.P. The IsdG-family of haem oxygenases degrades haem to a novel chromophore.Mol. Microbiol. 2010; 75: 1529-1538Crossref PubMed Scopus (107) Google Scholar) using either a Waters 2695 separation module with a Waters 2996 photodiode array detector or an Agilent Infinity 1260 multi-wavelength detector. With either setup the flow rate was 0.5 ml/min, and a Waters XTerra C18 column was used. Homologs of NWMN2274 were identified with BLASTP (35.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70322) Google Scholar) searches of the annotated genomes of S. aureus, Listeria monocytogenes, Bacillus subtilis, Bacillus anthracis, and Mycobacterium tuberculosis. Only hits with E-values equal to or less than 0.001 and covering 40% or more of the query sequence were considered significant. Multiple sequence alignments were generated with T-Coffee (36.Di Tommaso P. Moretti S. Xenarios I. Orobitg M. Montanyola A. Chang J.-M. Taly J.-F. Notredame C. T-Coffee. A web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension.Nucleic Acids Res. 2011; 39: W13-W17Crossref PubMed Scopus (731) Google Scholar, 37.Notredame C. Higgins D.G. Heringa J. T-coffee. A novel method for fast and accurate multiple sequence alignment.J. Mol. Biol. 2000; 302: 205-217Crossref PubMed Scopus (5446) Google Scholar). Maximum likelihood phylogenetic trees were built with the PhyML method in SeaView Version 4 (38.Gouy M. Guindon S. Gascuel O. SeaView version 4. A multiplatform graphical user interface for sequence alignment and phylogenetic tree building.Mol. Biol. Evol. 2010; 27: 221-224Crossref PubMed Scopus (4155) Google Scholar) using the LG model and bootstrapping with 100 replicates. The following S. aureus proteins (strain names in parentheses) all share >97% amino acid sequence identity to NWMN2274: SACOL2369 (SHY97–3906), SA2162 (N315), SAUSA300_2319 (USA300), SAOUHSC_02654 (NCTC 832), and MW2294 (MW2). Genes corresponding to these proteins were assumed to be orthologs of NWMN2274 in the analysis of microarray papers from various S. aureus strains. The heme-degrading proteins IsdG and IsdI require a source of electrons for porphyrin cleavage and iron release (22.Skaar E.P. Gaspar A.H. Schneewind O. IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus.J. Biol. 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• W1971624564 title "IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus" @default.
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