Abstract
In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split β-barrel conformation in each monomer comprising six β strands and three α helices. This structure resembles the split β-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split β-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.
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Acknowledgments
X-ray data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) beamline 22-ID at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This work was supported by American Heart Association Greater Southeast Affiliate Grant-in-Aid 15GRNT25600006 (ZE), Georgia State University Molecular Basis of Disease Area of Focus Seed Grant (ZE, ITW and GG), and Fellowship (AJS and MO), as well as National Science Foundation Grant CHE1506518 (to GG).
Funding
X-ray data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) beamline 22-ID at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This work was supported by American Heart Association Greater Southeast Affiliate Grant-in-Aid 15GRNT25600006 (ZE), Georgia State University Molecular Basis of Disease Area of Focus Seed Grant (ZE, ITW and GG), and Fellowship (AJS and MO), as well as National Science Foundation Grant CHE1506518 (to GG).
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Ankita J. Sachla and Mahamoudou Ouattara have contributed equally to this work.
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Fig. S1 HoloHupZ reaction product with and without acidification. UV–vis spectroscopic analysis of HupZ reaction performed with 10 μM of holoHupZ, CPR, and NADPH regeneration system for 5 h. The reaction mixture was acidified after 5 h with 10 mM HCl and partitioned into pyridine (33 %, dotted line). Inset depicts 10X resolution across 600–700 nm. Light blue line represents the reaction spectrum before the addition of acid; green line depicts measurements after acid addition; and red line indicates the spectrum in aqueous pyridine. Arrows indicate the increase or decrease in the spectral signatures. Supplementary material 1 (PDF 1057 kb)
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Sachla, A.J., Ouattara, M., Romero, E. et al. In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus. Biometals 29, 593–609 (2016). https://doi.org/10.1007/s10534-016-9937-1
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DOI: https://doi.org/10.1007/s10534-016-9937-1