Heme is essential for aerobic life and cellular respiration. The pathway by which eukaryotic cells make heme has been known for some time. Prokaryotic heme biosynthesis, by contrast, has been harder to describe. Recently, a pathway for heme biosynthesis that fills all the remaining gaps has been proposed for Gram- positive bacteria. This is a group of organisms that includes numerous important pathogens that are threats to public health and biodefense, such as the causative agents of MRSA, TB, anthrax, and plague. The pathway differs from the canonical one in its final three steps, with the greatest departure at its terminus. The last step is a double oxidative decarboxylation catalyzed by enzymes known as HemQs: a novel subtype of chlorite dismutases (Clds). The latter are heme enzymes that detoxify the chlorite end product of perchlorate respiration, converting it to Cl- to O2. The initial phase of this research resulted in a rigorous description of the structure, mechanism, and biology of O2-generating Clds from both perchlorate respirers and non-respiring pathogens. Leveraging the tools, insights, and scientific team assembled via work on Clds, this proposal aims at providing a description of HemQ function at the level of the individual molecule and extending to the cellular context. As preliminary work, a hemQ strain of Staphylococcus aureus has been generated and shown to be a heme auxotroph and small colony variant (SCV): a phenotype associated with intracellular persistence and antibiotic resistance. In tandem, the HemQ enzyme from S. aureus has been shown to oxidatively decarboxylate two of the four propionate side chains of coproheme III, in a reaction that depends strictly H2O2. Focusing on the S. aureus system, Aim 1 is to understand how HemQ binds and activates coproheme toward oxidative decarboxylation, producing structural and energetic models of SaHemQ in complex with its substrate (coproheme III), intermediate (harderoheme) and product (heme b).
Aim 2 is to test a mechanism for HemQ's reaction, in which coproheme is both substrate and cofactor in the peroxidation. Time-resolved and kinetic isotope methods will be used to examine a series of hypotheses invoking a ferric-hydroperoxy intermediate and intramolecular hydrogen atom transfer. Finally, aim 3 uses genetic, cell-based, and biochemical methods to understand HemQ's function in the context of the cell and evolution. We expect completion of the proposed work to define the ultimate step of a pathway that is absolutely fundamental to aerobic life, essential for robust pathogenic growth, and clinically connected to the development of persistence and antibiotic resistance.

Public Health Relevance

Heme is essential for robust bacterial growth in air. HemQs are enzymes that were just discovered to catalyze the ultimate step in heme biosynthesis in Gram-positive bacteria, a group containing the causative agents of tuberculosis, MRSA infection, listeriosis, anthrax, and other diseases that are major threats to public health. The gol of the proposed research is to understand how HemQ works, alone and in its cellular context.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM090260-07A1
Application #
8964883
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2009-08-01
Project End
2019-06-30
Budget Start
2015-09-05
Budget End
2016-08-31
Support Year
7
Fiscal Year
2015
Total Cost
$304,432
Indirect Cost
$64,432
Name
Montana State University - Bozeman
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
625447982
City
Bozeman
State
MT
Country
United States
Zip Code
59717
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Geeraerts, Zachary; Celis, Arianna I; Mayfield, Jeffery A et al. (2018) Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes. Biochemistry 57:1501-1516
Choby, Jacob E; Grunenwald, Caroline M; Celis, Arianna I et al. (2018) Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis. MBio 9:
Celis, Arianna I; Gauss, George H; Streit, Bennett R et al. (2017) Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ). J Am Chem Soc 139:1900-1911
Geeraerts, Zachary; Rodgers, Kenton R; DuBois, Jennifer L et al. (2017) Active Sites of O2-Evolving Chlorite Dismutases Probed by Halides and Hydroxides and New Iron-Ligand Vibrational Correlations. Biochemistry 56:4509-4524
Streit, Bennett R; Celis, Arianna I; Shisler, Krista et al. (2017) Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b. Biochemistry 56:189-201
Streit, Bennett R; Kant, Ravi; Tokmina-Lukaszewska, Monika et al. (2016) Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway. J Biol Chem 291:862-71
Machovina, Melodie M; Usselman, Robert J; DuBois, Jennifer L (2016) Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations. J Biol Chem 291:17816-28
Celis, Arianna I; DuBois, Jennifer L (2015) Substrate, product, and cofactor: The extraordinarily flexible relationship between the CDE superfamily and heme. Arch Biochem Biophys 574:3-17
DuBois, Jennifer L; Ojha, Sunil (2015) Production of dioxygen in the dark: dismutases of oxyanions. Met Ions Life Sci 15:45-87

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