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Radical New Paradigm for Heme Degradation in Escherichia Coli O157:H7

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Radical New Paradigm for Heme Degradation in Escherichia Coli O157:H7 Radical new paradigm for heme degradation in Escherichia coli O157:H7 Joseph W. LaMattinaa,b, David B. Nixa,c, and William Nicholas Lanzilottaa,b,1 aDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; bCenter for Metalloenzyme Studies, University of Georgia, Athens, GA 30602; and cComplex Carbohydrate Research Center, University of Georgia, Athens, GA 30602 Edited by Iqbal Hamza, University of Maryland, College Park, MD, and accepted by Editorial Board Member Michael A. Marletta September 7, 2016 (received for review February 25, 2016) All of the heme-degrading enzymes that have been characterized Vibrio cholerae and Escherichia coli O157:H7 are hemolytic en- to date require molecular oxygen as a cosubstrate. Escherichia coli teric pathogens that colonize the nonsterile region of the lower O157:H7 has been shown to express heme uptake and transport intestines. Despite their ability to use heme as an iron source, a proteins, as well as use heme as an iron source. This enteric pathogen bona fide heme oxygenase has not been characterized for either colonizes the anaerobic space of the lower intestine in mammals, yet organism. However, both organisms contain a heme uptake operon no mechanism for anaerobic heme degradation has been reported. that is up-regulated during iron duress (14, 15). The operon en- Herein we provide evidence for an oxygen-independent heme- codes for the heme uptake and transport machinery, as well as degradation pathway. Specifically, we demonstrate that ChuW is three additional genes, chuW, chuX,andchuY. (Fig. 1A)(16). a radical S-adenosylmethionine methyltransferase that catalyzes a ChuW is annotated as a member of the radical S-adenosylme- radical-mediated mechanism facilitating iron liberation and the thionine (SAM) superfamily, a large class of enzymes that perform “ ” production of the tetrapyrrole product we termed anaerobilin. a wide array of difficult chemical reactions (17, 18). Enzymes be- We further demonstrate that anaerobilin can be used as a sub- longing to the radical SAM superfamily share common cofactor strate by ChuY, an enzyme that is coexpressed with ChuW in vivo requirements and mechanistic features, such as the coordination of along with the heme uptake machinery. Our findings are discussed in a redox active [4Fe-4S] cluster ligated by three protein-derived terms of the competitive advantage this system provides for enteric cysteine residues, often located within a CX3CX2C motif. This bacteria, particularly those that inhabit an anaerobic niche in the coordination environment facilitates the interaction of SAM at the intestines. unique iron site of the cluster. In the reduced state (formally 1+), the [4Fe-4S] cluster reductively cleaves SAM, forming a highly heme | pathogen | anaerobic | intestinal microbiome | radical SAM oxidative 5′-deoxyadenosyl-5′-radical (5′-dA•) that is subsequently used for catalysis. ChuW was originally annotated as the radical ron acquisition is essential to the survival of all cellular or- SAM enzyme HemN, an anaerobic coproporphyrinogen oxi- Iganisms and is a major barrier for a microorganism during dase. HemN is involved in the anaerobic biosynthesis of heme, pathogenesis of a mammalian host (1). For example, an enteric performing two subsequent decarboxylation reactions to pro- pathogen entering through the digestive tract must compete with duce protoporphyrinogen IX (19). However, previous work has the host, as well as other intestinal microflora for iron. Interestingly, shown that ChuW homologs do not retain the essential func- the most abundant source of dietary iron is found in heme (2), a tional motifs, nor do they rescue a HemN KO in Salmonella molecule that is also responsible for essential cellular processes by enterica, and thus likely catalyze a different reaction (14, 20). A serving as a cofactor in a variety of enzymes (3). The degradation of role in heme biosynthesis is also inconsistent with the location of heme also plays an important role in iron homeostasis and cell chuW in an iron-regulated operon adjacent to genes that are signaling in cyanobacteria, plants, and mammals (1, 4). Considering required for heme uptake (Fig. 1). the bioavailability of dietary heme and the importance of iron as Sequence alignments indicate that ChuW has homology to the a nutrient, it is not surprising that pathways have evolved in radical SAM methyltransferases (RSMTs). RSMTs are a sub- pathogenic bacteria to transport and degrade heme for the sole family of radical SAM enzymes that function in the transfer of purpose of iron acquisition (5). Furthermore, the ability to accom- plish the liberation of iron from heme under strictly anaerobic con- Significance ditions would be advantageous to the enteric bacteria that can inhabit certain niches of the intestines. The ability of pathogenic microorganisms to acquire iron from Enzymes that catalyze the opening of the porphyrin ring have heme has been shown to be a valid antimicrobial target. How- been well characterized and are collectively referred to as heme ever, all of the known mechanisms for heme catabolism that lead oxygenases (6). This classification is based on the common mech- to iron release in bacteria and higher eukaryotes are dependent anistic property of activating molecular oxygen by a “P450-like” on molecular oxygen as a cosubstrate. The human gut is domi- mechanism to catalyze the oxidativedegradationofthehemeco- nated by strictly anaerobic bacteria, and in this work, we provide factor (7–9). Recently, two heme oxygenases have been character- evidence for anaerobic heme degradation in enteric pathogens. ized in the organisms Staphylococcus aureus and Mycobacterium The anaerobic mechanism is significantly different from what has tuberculosis that degrade heme to the unique chromophores been observed in other bacteria and may provide a unique op- staphylobilin (10) and mycobilin (11), respectively. Although portunity for a new class of antimicrobial compounds. molecular oxygen is still required, neither of these degradation Author contributions: W.N.L. designed research; J.W.L. and D.B.N. performed research; mechanisms result in carbon monoxide production, presumably J.W.L., D.B.N., and W.N.L. analyzed data; and J.W.L. and W.N.L. wrote the paper. due to the pathophysiology of these organisms (12). These re- The authors declare no conflict of interest. ports significantly advance the hypothesis that organisms evolved This article is a PNAS Direct Submission. I.H. is a Guest Editor invited by the Editorial a variety of heme degradation mechanisms to satisfy their own Board. physiological needs (13), thus raising the following question: how 1To whom correspondence should be addressed. Email: [email protected]. do hemolytic organisms use heme as an iron source during in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fection or colonization in an anaerobic environment? 1073/pnas.1603209113/-/DCSupplemental. 12138–12143 | PNAS | October 25, 2016 | vol. 113 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1603209113 Downloaded by guest on September 26, 2021 Flavodoxin Is the Electron Source for the Radical SAM Enzyme ChuW. A Anaerobilin Anaerobilin EC3742 Synthase Reductase EC3749 In addition to being a radical SAM enzyme, sequence analysis ChuS ChuA ChuT ChuW ChuX ChuY ChuU HmuV indicates that ChuW is distinct from HemN and more similar to Outer Periplasmic Unknown Cytoplasmic ABC Transporter the class C RSMTs (Fig. S1). Therefore, we looked for both Storage membrane Shuttle Function Receptor 5′-deoxyadenosine (5′-dA) and S-adenosylhomocysteine (SAH) B 1.0 C production during ChuW turnover by HPLC. In our assays, we i. 1.5 ox 0.8 used the heme analog deuteroheme as the substrate due to the 2.04 0.6 insolubility of hemin. Consistent with what others have reported, 0.4 1.92 1.0 ii. we observed “abortive cleavage” of SAM to 5′-dA in the absence Relative ChuW 0.2 of deuteroheme when using the chemical reductant sodium 0.0 Absorbance 020406080 A 0.5 Ti 3+ -citrate (μM) dithionite (Fig. S2 ). Minimal SAH production was observed Ox when deuteroheme was absent and the chemical reductant was 3000 3200 3400 3600 3800 4000 Red 0.0 Magnetic Field (Gauss) used (Fig. S2B). However, when using the E. coli flavodoxin 300 400 500 600 Wavelength (nm) (EcFldA)/ferredoxin (flavodoxin):NADP+ oxidoreductase (EcFpr)/ NADPH system (26), abortive cleavage was not observed, and both Fig. 1. Genetic organization of the heme utilization operon in E. coli O157: 5′-dA and SAH were produced at 1:1 stoichiometries only when H7 and characterization of the Fe-S cluster in purified ChuW. (A) ChuW is substrate was present (Fig. S2C, trace ii). These observations genetically adjacent to the heme uptake machinery that is expressed during iron starvation. (B) UV-visible spectra of isolated ChuW and reduction of the further underscore the importance of using a physiological elec- + Fe-S cluster. Scans were recorded after injecting with equivalents of Ti3 tron source for the in vitro characterization of radical SAM en- citrate. (Inset) Percentage of oxidized ChuW based on the relative change in zymes (26–28). Therefore, we used the physiological electron the adsorption at 400 nm plotted with respect to the concentration of Ti3+ donor system in place of a chemical reductant. Quantitation of citrate added. (C) EPR spectra of ChuW in the absence and presence of 1 mM the total iron that was subject to chelation (labile iron) was also sodium dithionite (top and bottom trace, respectively). Spectra are a result performed. Measurements of the labile iron were performed at of three scans recorded at 10 K with 1-MW microwave power, a modulation the start of the reaction and after 30 min. It should be recognized frequency of 100 kHz, and modulation amplitude of 6.477 gauss (G). that the catalytic cluster is fragile and also subject to chelation in contrast to iron bound to the porphyrin. Notably, we observe an increase in the labile iron during ChuW turnover; specifically, we methyl groups to unreactive carbon atoms and, in some cases, ± μ promote significant chemical rearrangements (21, 22).
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