Publications

• Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin.

  Dailey H.A., Gerdes S., Dailey T.A., Burch J.S., Phillips J.D.

  It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, ... >> More

  It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, they oxidize coproporphyrinogen to coproporphyrin, insert ferrous iron to make Fe-coproporphyrin (coproheme), and then decarboxylate coproheme to generate protoheme. This pathway is specified by three genes named hemY, hemH, and hemQ. The analysis of 982 representative prokaryotic genomes is consistent with this pathway being the most ancient heme synthesis pathway in the Eubacteria. Our results identifying a previously unknown branch of tetrapyrrole synthesis support a significant shift from current models for the evolution of bacterial heme and chlorophyll synthesis. Because some organisms that possess this coproporphyrin-dependent branch are major causes of human disease, HemQ is a novel pharmacological target of significant therapeutic relevance, particularly given high rates of antimicrobial resistance among these pathogens. << Less

  Proc. Natl. Acad. Sci. U.S.A. 112:2210-2215(2015) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (HemQ).

  Celis A.I., Gauss G.H., Streit B.R., Shisler K., Moraski G.C., Rodgers K.R., Lukat-Rodgers G.S., Peters J.W., DuBois J.L.

  Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H₂O₂ as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution ... >> More

  Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H₂O₂ as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H₂O₂-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr^•. This residue then abstracts a net hydrogen atom (H^•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO₂ products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e^-/H⁺ transfers and a novel route for the conversion of carboxylic acids to alkenes. << Less

  J. Am. Chem. Soc. 139:1900-1911(2017) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The HemQ coprohaem decarboxylase generates reactive oxygen species: implications for the evolution of classical haem biosynthesis.

  Hobbs C., Dailey H.A., Shepherd M.

  Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes, including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates, including non-metallated porphyrins, such as protoporphyrin IX, which is well ... >> More

  Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes, including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates, including non-metallated porphyrins, such as protoporphyrin IX, which is well known to generate reactive oxygen species in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin. Here, we demonstrate, for the first time, that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyrin III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporphyrin IX (but not coproporphyrin III). Assays with hydrogen peroxide, horseradish peroxidase, superoxide dismutase, and catalase confirm that this stimulatory effect is mediated by superoxide. Structural modelling reveals that HemQ enzymes do not possess the structural attributes that are common to peroxidases that form compound I [Fe^IV==O]⁺, which taken together with the superoxide data leaves Fenton chemistry as a likely route for the superoxide-mediated stimulation of protoporphyrinogen IX oxidase activity of HemY. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. This work has implications for the divergent evolution of haem biosynthesis in ancestral microorganisms, and provides new structural and mechanistic insights into a recently discovered oxidative decarboxylase reaction. << Less

  Biochem. J. 473:3997-4009(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Reactions of ferrous coproheme decarboxylase (HemQ) with O2 and H2O2 yield ferric heme b.

  Streit B.R., Celis A.I., Shisler K., Rodgers K.R., Lukat-Rodgers G.S., DuBois J.L.

  A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O₂ and H₂O₂ are available as cellular oxida ... >> More

  A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O₂ and H₂O₂ are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O₂ to the ferric state. The subsequent second-order reaction between the ferric complex and H₂O₂ is slow, pH-dependent, and further decelerated by D₂O₂ (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H₂O₂. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H₂O₂ cleavage is therefore unclear. From a cellular perspective, the use of H₂O₂ as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis. << Less

  Biochemistry 56:189-201(2017) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• From chlorite dismutase towards HemQ - the role of the proximal H-bonding network in haeme binding.

  Hofbauer S., Howes B.D., Flego N., Pirker K.F., Schaffner I., Mlynek G., Djinovic-Carugo K., Furtmuller P.G., Smulevich G., Obinger C.

  Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positi ... >> More

  Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from 'Candidatus Nitrospira defluvii' (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV-vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure-function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria. << Less

  Biosci Rep 36:e00312-e00312(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Discovery and characterization of HemQ: an essential heme biosynthetic pathway component.

  Dailey T.A., Boynton T.O., Albetel A.N., Gerdes S., Johnson M.K., Dailey H.A.

  Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high ... >> More

  Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 10(4) min(-1). We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis. << Less

  J. Biol. Chem. 285:25978-25986(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria.

  Dailey H.A., Gerdes S.

  Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named ... >> More

  Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are iron-coproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. The heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed. Thus, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis. << Less

  Arch. Biochem. Biophys. 574:27-35(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Unusual peroxide-dependent, heme-transforming reaction catalyzed by HemQ.

  Celis A.I., Streit B.R., Moraski G.C., Kant R., Lash T.D., Lukat-Rodgers G.S., Rodgers K.R., DuBois J.L.

  A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from ... >> More

  A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out. << Less

  Biochemistry 54:4022-4032(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ-lessons from the first crystal structure and kinetic studies.

  Hofbauer S., Mlynek G., Milazzo L., Puhringer D., Maresch D., Schaffner I., Furtmuller P.G., Smulevich G., Djinovic-Carugo K., Obinger C.

  Heme biosynthesis in Gram-positive bacteria follows a recently described coproporphyrin-dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme-HemQ from Listeria monocytogenes) at 1.69 Å res ... >> More

  Heme biosynthesis in Gram-positive bacteria follows a recently described coproporphyrin-dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme-HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV-vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five-coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H-bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H₂ O₂ molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (k_cat /K_M = 1.8 × 10² m^-1 ·s^-1 at pH 7.0) and occurs in a stepwise manner with a three-propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two-step reaction mechanism. Furthermore, the structure of coproheme-HemQ is compared to that of the phylogenetically related heme b-containing chlorite dismutases.<h4>Database</h4>Structural data are available in the PDB under the accession number 5LOQ. << Less

  FEBS J. 283:4386-4401(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.