DOCSLIB.ORG

Human Oxygen Sensing May Have Origins in Prokaryotic Elongation Factor Tu Prolyl-Hydroxylation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Human Oxygen Sensing May Have Origins in Prokaryotic Elongation Factor Tu Prolyl-Hydroxylation Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation John S. Scottia, Ivanhoe K. H. Leunga,1,2, Wei Gea,b,1, Michael A. Bentleyc, Jordi Papsd, Holger B. Kramere, Joongoo Leea, WeiShen Aika, Hwanho Choia, Steinar M. Paulsenc,3, Lesley A. H. Bowmanf, Nikita D. Loika,4, Shoichiro Horitaa,e, Chia-hua Hoa,5, Nadia J. Kershawa,6, Christoph M. Tangf, Timothy D. W. Claridgea, Gail M. Prestonc, Michael A. McDonougha, and Christopher J. Schofielda,7 aChemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; bChinese Academy of Medical Sciences, Beijing 100005, China; cDepartment of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom; dDepartment of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom; eDepartment of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; and fDepartment of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom Edited by Gregg L. Semenza, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 5, 2014 (received for review May 30, 2014) The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases Results in eukaryotes include collagen stabilization, hypoxia sensing, and Pseudomonas spp. Contain a Functional PHD. To investigate the role translational regulation. The hypoxia-inducible factor (HIF) sensing of a putative PHD homolog in Pseudomonas aeruginosa (PPHD), system is conserved in animals, but not in other organisms. How- we initially characterized a PPHD insertional mutant strain. ever, bioinformatics imply that 2OG-dependent prolyl-hydroxy- Metabolic screening studies revealed that the PPHD mutant strain lases (PHDs) homologous to those acting as sensing components displays impaired growth in the presence of iron chelators (e.g., for the HIF system in animals occur in prokaryotes. We report 2,2-bipyridine) and produces increased levels of the bacterial vir- cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein ulence factor pyocyanin (SI Appendix,Fig.S1)(12),implying (PPHD) contain a 2OG oxygenase related in structure and function PPHD is involved in iron regulation. Ferrous iron is essential for to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout PHD catalysis and treatment of human cells with iron chelators, mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We Significance identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal The Fe(II)- and 2-oxoglutarate (2OG)-dependent hypoxia- structure of PPHD reveals striking similarity to human PHD2 and inducible transcription factor prolyl-hydroxylases play a central a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal struc- role in human oxygen sensing and are related to other prolyl- ture of PPHD complexed with intact EF-Tu reveals that major con- hydroxylases involved in eukaryotic collagen biosynthesis and formational changes occur in both PPHD and EF-Tu, including ribosomal modification. The finding that a PHD-related prolyl-hy- > a 20-Å movement of the EF-Tu switch I loop. Comparison of droxylase in Pseudomonas spp. regulates pyocyanin biosynthesis the PPHD structures with those of HIF and collagen PHDs reveals supports prokaryotic origins for the eukaryotic prolyl-hydrox- conservation in substrate recognition despite diverse biological ylases. The identification of the switch I loop of elongation factor roles and origins. The observed changes will be useful in designing Tu (EF-Tu) as a Pseudomonas prolyl-hydroxylase domain contain- new types of 2OG oxygenase inhibitors based on various confor- ing protein (PPHD) substrate provides evidence of roles for 2OG mational states, rather than active site iron chelators, which make oxygenases in both translational and transcriptional regulation. A up most reported 2OG oxygenase inhibitors. Structurally in- structure of the PPHD:EF-Tu complex, the first to the authors’ BIOCHEMISTRY formed phylogenetic analyses suggest that the role of prolyl- knowledge of a 2OG oxygenase with its intact protein substrate, hydroxylation in human hypoxia sensing has ancient origins. reveals that major conformational changes occur in both PPHD and EF-Tu and will be useful in the design of new prolyl- he hypoxia-inducible transcription factor (HIF) is a major hydroxylase inhibitors. Tregulator of the response to limited oxygen availability in humans and other animals (1–3). A hypoxia-sensing component of Author contributions: J.S.S., I.K.H.L., W.G., G.M.P., M.A.M., and C.J.S. designed research; J.S.S., I.K.H.L., W.G., M.A.B., J.P., H.B.K., J.L., W.A., H.C., S.M.P., L.A.H.B., N.D.L., C.-h.H., and the HIF system is provided by 2-oxoglutarate (2OG)-dependent N.J.K. performed research; J.S.S. contributed new reagents/analytic tools; J.S.S., I.K.H.L., W.G., and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydrox- M.A.B., J.P., H.B.K., C.M.T., T.D.W.C., G.M.P., M.A.M., and C.J.S. analyzed data; and J.S.S., S.H., ylation of HIF-α subunits, a posttranslational modification that M.A.M., and C.J.S. wrote the paper. enhances binding of HIF-α to the von Hippel-Lindau protein The authors declare no conflict of interest. (pVHL), so targeting HIF-α for proteasomal degradation. The Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG www.pdb.org (PDB ID codes 4J25, 4J0Q, 4IW3), and sequence data deposited in Uniprot, www.uniprot.org/uniprot (accession nos. Q88CM1 and Q9I6I1) and GenBank, www.ncbi. oxygenases that catalyze prolyl-hydroxylation, which also includes nih.gov/protein (accession nos. AAN70724.1 and AAG03699.1). the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydrox- 1I.K.H.L. and W.G. contributed equally to this work. ylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases 2 Present address: School of Chemical Sciences, University of Auckland, Auckland 1142, include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), New Zealand. which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many 3Present address: Department of Medical Biology, University of Tromsø, N-9019 Tromsø, Norway. eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hy- 4Present address: Department of Chemistry, University of Tokyo, 113-0033 Tokyo, Japan. – droxylation of Skp1, a ubiquitin ligase subunit (5 9). The HIF- 5Present address: Helmholtz Zentrum Munchen, 85765 Munchen, Germany. PHD-VHL triad is likely present in all animals, but probably 6Present address: The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, not in other organisms (3). However, structurally informed Australia. bioinformatic analyses imply the presence of PHD homologs in 7To whom correspondence should be addressed. Email: [email protected]. bacteria (10, 11), including in Pseudomonas spp, suggesting This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PHDs may have ancient origins. 1073/pnas.1409916111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1409916111 PNAS | September 16, 2014 | vol. 111 | no. 37 | 13331–13336 Downloaded by guest on October 1, 2021 including pyocyanin (13), and causes HIF-α up-regulation via to reflect its role as a hypoxia sensor, these preliminary differ- PHD inhibition, suggesting related roles for PPHD and the animal ences are of interest. In particular, the higher Km for Fe(II) is PHDs (1). notable given the phenotypic results on the PPHD insertional mutant strain, supporting a role for PPHD in iron regulation. Elongation Factor Tu Is a PPHD Substrate. We then investigated The switch I loop of EF-Tu plays a central role in bacterial possible PPHD substrates. Mass spectrometric (MS) studies translation by coupling GTP hydrolysis with conformational identified elongation factor Tu (EF-Tu) as a PPHD binding changes from the GTP-bound active state to the GDP-bound partner and potential substrate in Pseudomonas putida (SI Ap- inactive state (21, 22). NMR was used to directly monitor pendix,TableS3). This assignment is supported by a proteomic kirromycin-induced GTP hydrolysis by EF-Tuputida; we observed MS study on Shewanella oneidensis, reporting that EF-Tu is mod- no difference in the GTP hydrolysis rate for Hyp54 EF-Tu (0.71 ± ified by a +16-Da mass shift (14); bioinformatics reveal Shewanella 0.02 μM/min) and unmodified EF-Tu (0.76 ± 0.08 μM/min; – oneidensis contains a potential hydroxylase closely related to SI Appendix,Fig.S5A). However, addition of PPHDputida to the EF PPHD (38% identity). We tested a set of 14 synthetic peptides Tu GTP hydrolysis reaction decreased the rate of GTP hydrolysis covering the 19 prolines in the EF-Tu sequence as substrates for in a metal ion and 2OG-dependent manner [Zn(II) 0.60 ± 0.05 μ ± μ isolated recombinant PPHDputida (SI Appendix, Table S4). We M/min; Zn(II) and 2OG 0.49 0.16 M/min; Zn(II) was used as identified an EF-Tu peptide as undergoing a +16-Da mass shift a surrogate for Fe(II) and a PPHDputida inhibitor; SI Appendix, – (Fig. 1B and SI Appendix, Table S4); MSMS analyses demon- Fig. S5 B D]. In P. aeruginosa, we observed no difference in the strated hydroxylation at Pro54 (Hyp54; SI Appendix, Fig. S2A). global translation rate between the PPHD mutant strain and the wild-type under standard growth conditions, as measured by Amino acid analyses reveal that PPHDputida catalyzes trans 35 prolyl-4-hydroxylation of EF-Tu, as occurs for PHD-catalyzed [ S]methionine incorporation (SI Appendix,Fig.S5E). The effect of PPHDputida on the GTP hydrolysis rate is less with hydroxy- HIF and CP4H-catalyzed collagen prolyl-hydroxylation (SI Ap- ± μ pendix, Fig.
Load more

Recommended publications
  • Regulation of Ribulose-1, 5-Bisphosphate Carboxylase
    Plant Physiol. (1993) 102: 21-28 Regulation of Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase Activity in Response to Reduced Light lntensity in C4 Plants' Rowan F. Sage* and Jeffrey R. Seemann Department of Botany, University of Georgia, Athens, Ceorgia 30602 (R.F.S.); and Department of Biochemistry, University of Nevada, Reno, Nevada 89557 (J.R.S.) tion of Rubisco activity by reversible carbamylation occurs in lhe light-dependent regulation of ribulose-1,5-bisphosphate response to changes in light intensity as well as the concen- carboxylase/oxygenase (Rubisco) activity was studied in 16 species tration of C02and 02,whereas inhibition of Rubisco activity of C, plants representing all three biochemical subtypes and a by CAlP occurs only in response to varying PPFD (Sharkey variety of taxonomic groups. Rubisco regulation was assessed by et al., 1986; Sage et al., 1990; Seemann et al., 1990). At measuring (a) the ratio of initial to total Rubisco activity, which physiological levels of COz in C3 plants (5-10 PM), full reflects primarily the carbamylation state of the enzyme, and (b) carbamylation of Rubisco requires Rubisco activase (Salvucci, total Rubisco activity per mo1 of Rubisco catalytic sites, which 1989; Portis, 1990). In the absence of Rubisco activase, only declines when 2-carboxyarabinitol I-phosphate (CAlP) binds to carbamylated Rubisco. In all species examined, the activity ratio of 20 to 40% of Rubisco catalytic sites are carbamylated under Rubisco declined with a reduction in light intensity, although sub- physiological conditions, leading to a significant inhibition of stantial variation was apparent between species in the degree of photosynthesis (Salvucci, 1989; Portis, 1990).
    [Show full text]
  • Hypoxia and Oxygen-Sensing Signaling in Gene Regulation and Cancer Progression
    International Journal of Molecular Sciences Review Hypoxia and Oxygen-Sensing Signaling in Gene Regulation and Cancer Progression Guang Yang, Rachel Shi and Qing Zhang * Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; [email protected] (G.Y.); [email protected] (R.S.) * Correspondence: [email protected]; Tel.: +1-214-645-4671 Received: 6 October 2020; Accepted: 29 October 2020; Published: 31 October 2020 Abstract: Oxygen homeostasis regulation is the most fundamental cellular process for adjusting physiological oxygen variations, and its irregularity leads to various human diseases, including cancer. Hypoxia is closely associated with cancer development, and hypoxia/oxygen-sensing signaling plays critical roles in the modulation of cancer progression. The key molecules of the hypoxia/oxygen-sensing signaling include the transcriptional regulator hypoxia-inducible factor (HIF) which widely controls oxygen responsive genes, the central members of the 2-oxoglutarate (2-OG)-dependent dioxygenases, such as prolyl hydroxylase (PHD or EglN), and an E3 ubiquitin ligase component for HIF degeneration called von Hippel–Lindau (encoding protein pVHL). In this review, we summarize the current knowledge about the canonical hypoxia signaling, HIF transcription factors, and pVHL. In addition, the role of 2-OG-dependent enzymes, such as DNA/RNA-modifying enzymes, JmjC domain-containing enzymes, and prolyl hydroxylases, in gene regulation of cancer progression, is specifically reviewed. We also discuss the therapeutic advancement of targeting hypoxia and oxygen sensing pathways in cancer. Keywords: hypoxia; PHDs; TETs; JmjCs; HIFs 1. Introduction Molecular oxygen serves as a co-factor in many biochemical processes and is fundamental for aerobic organisms to maintain intracellular ATP levels [1,2].
    [Show full text]
  • Distinct RNA N-Demethylation Pathways Catalyzed by Nonheme Iron ALKBH5 and FTO Enzymes Enable Regulation of Formaldehyde Release Rates
    Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates Joel D. W. Toha,1, Steven W. M. Crossleya,1, Kevin J. Bruemmera, Eva J. Gea, Dan Hea, Diana A. Iovana, and Christopher J. Changa,b,2 aDepartment of Chemistry, University of California, Berkeley, CA 94720; and bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Edited by Amy C. Rosenzweig, Northwestern University, Evanston, IL, and approved August 24, 2020 (received for review April 17, 2020) The AlkB family of nonheme Fe(II)/2-oxoglutarate–dependent oxy- m6A (23) is relatively stable compared to other hydroxymethyl- genases are essential regulators of RNA epigenetics by serving as containing nucleobases and has been reported to decay to the free erasers of one-carbon marks on RNA with release of formaldehyde adenosine (A) base over 10 h (22). m6A is the most prominent (FA). Two major human AlkB family members, FTO and ALKBH5, modification of messenger RNA (mRNA) (24, 25) and is installed by both act as oxidative demethylases of N6-methyladenosine (m6A) the S-adenosylmethionine–dependent METTL3/14-WTAP writer but furnish different major products, N6-hydroxymethyladenosine complex (26) and removed by two human AlkB eraser enzymes, fat (hm6A) and adenosine (A), respectively. Here we identify founda- mass and obesity-associated protein (FTO) (4) and AlkB family tional mechanistic differences between FTO and ALKBH5 that pro- member 5 (ALKBH5) (27). Internal m6A modifications control the mote these distinct biochemical outcomes. In contrast to FTO, fate of mRNA (28–32) through translation, splicing, localization, which follows a traditional oxidative N-demethylation pathway stability, and decay and are connected to cancer progression, im- to catalyze conversion of m6A to hm6A with subsequent slow mune responses, and metabolic states (27, 29, 32–36).
    [Show full text]
  • Potential for and Distribution of Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms
    materials Communication Potential for and Distribution of Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms Liyuan Hou and Erica L.-W. Majumder * Department of Chemistry, SUNY College of Environmental Science and Forestry, Syracuse, NY 13210, USA; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +1-3154706854 Abstract: Polystyrene (PS) is one of the main polymer types of plastic wastes and is known to be resistant to biodegradation, resulting in PS waste persistence in the environment. Although previous studies have reported that some microorganisms can degrade PS, enzymes and mechanisms of microorganism PS biodegradation are still unknown. In this study, we summarized microbial species that have been identified to degrade PS. By screening the available genome information of microorganisms that have been reported to degrade PS for enzymes with functional potential to depolymerize PS, we predicted target PS-degrading enzymes. We found that cytochrome P4500s, alkane hydroxylases and monooxygenases ranked as the top potential enzyme classes that can degrade PS since they can break C–C bonds. Ring-hydroxylating dioxygenases may be able to break the side-chain of PS and oxidize the aromatic ring compounds generated from the decomposition of PS. These target enzymes were distributed in Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, suggesting a broad potential for PS biodegradation in various earth environments and microbiomes. Our results provide insight into the enzymatic degradation of PS and suggestions for realizing the biodegradation of this recalcitrant plastic. Citation: Hou, L.; Majumder, E.L. Keywords: plastics; polystyrene biodegradation; enzymatic biodegradation; monooxygenase; alkane Potential for and Distribution of hydroxylase; cytochrome P450 Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms.
    [Show full text]
  • Dn056490.Pdf
    A Rapid Transcriptome Response Is Associated with Desiccation Resistance in Aerially-Exposed Killifish Embryos Ange`le Tingaud-Sequeira1¤a, Juan-Jose´ Lozano2, Cinta Zapater1, David Otero1, Michael Kube3¤b, Richard Reinhardt3¤c, Joan Cerda` 1* 1 Institut de Recerca i Tecnologia Agroalimenta`ries (IRTA)-Institut de Cie`ncies del Mar, CSIC, Barcelona, Spain, 2 Bioinformatics Platform, CIBERHED, Barcelona, Spain, 3 Max Planck Institute for Molecular Genetics, Berlin-Dahlem, Germany Abstract Delayed hatching is a form of dormancy evolved in some amphibian and fish embryos to cope with environmental conditions transiently hostile to the survival of hatchlings or larvae. While diapause and cryptobiosis have been extensively studied in several animals, very little is known concerning the molecular mechanisms involved in the sensing and response of fish embryos to environmental cues. Embryos of the euryhaline killifish Fundulus heteroclitus advance dvelopment when exposed to air but hatching is suspended until flooding with seawater. Here, we investigated how transcriptome regulation underpins this adaptive response by examining changes in gene expression profiles of aerially incubated killifish embryos at ,100% relative humidity, compared to embryos continuously flooded in water. The results confirm that mid-gastrula embryos are able to stimulate development in response to aerial incubation, which is accompanied by the differential expression of at least 806 distinct genes during a 24 h period. Most of these genes (,70%) appear to be differentially expressed within 3 h of aerial exposure, suggesting a broad and rapid transcriptomic response. This response seems to include an early sensing phase, which overlaps with a tissue remodeling and activation of embryonic development phase involving many regulatory and metabolic pathways.
    [Show full text]
  • Somatic Aging Pathways Regulate Reproductive Plasticity in Caenorhabditis Elegans Maria C Ow, Alexandra M Nichitean, Sarah E Hall*
    RESEARCH ARTICLE Somatic aging pathways regulate reproductive plasticity in Caenorhabditis elegans Maria C Ow, Alexandra M Nichitean, Sarah E Hall* Department of Biology, Syracuse University, Syracuse, United States Abstract In animals, early-life stress can result in programmed changes in gene expression that can affect their adult phenotype. In C. elegans nematodes, starvation during the first larval stage promotes entry into a stress-resistant dauer stage until environmental conditions improve. Adults that have experienced dauer (postdauers) retain a memory of early-life starvation that results in gene expression changes and reduced fecundity. Here, we show that the endocrine pathways attributed to the regulation of somatic aging in C. elegans adults lacking a functional germline also regulate the reproductive phenotypes of postdauer adults that experienced early-life starvation. We demonstrate that postdauer adults reallocate fat to benefit progeny at the expense of the parental somatic fat reservoir and exhibit increased longevity compared to controls. Our results also show that the modification of somatic fat stores due to parental starvation memory is inherited in the F1 generation and may be the result of crosstalk between somatic and reproductive tissues mediated by the germline nuclear RNAi pathway. Introduction Evidence indicating that experiences during early development affect behavior and physiology in a stress-specific manner later in life is abundant throughout the animal kingdom (Telang and Wells, *For correspondence: [email protected] 2004; Weaver et al., 2004; Binder et al., 2008; Pellegroms et al., 2009; van Abeelen et al., 2012; Zhao and Zhu, 2014; Canario et al., 2017; Dantzer et al., 2019; Vitikainen et al., 2019).
    [Show full text]
  • A Three-Component Dicamba O-Demethylase from Pseudomonas Maltophilia, Strain DI-6 GENE ISOLATION, CHARACTERIZATION, and HETEROLOGOUS EXPRESSION*
    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 26, Issue of July 1, pp. 24759–24767, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Three-component Dicamba O-Demethylase from Pseudomonas maltophilia, Strain DI-6 GENE ISOLATION, CHARACTERIZATION, AND HETEROLOGOUS EXPRESSION* Received for publication, January 18, 2005, and in revised form, March 16, 2005 Published, JBC Papers in Press, April 26, 2005, DOI 10.1074/jbc.M500597200 Patricia L. Herman‡, Mark Behrens, Sarbani Chakraborty, Brenda M. Chrastil, Joseph Barycki, and Donald P. Weeks§ From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 65888-0664 Dicamba O-demethylase is a multicomponent enzyme The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) from Pseudomonas maltophilia, strain DI-6, that cata- has been used to effectively control broadleaf weeds in crops lyzes the conversion of the widely used herbicide di- such as corn and wheat for almost 40 years. Like a number of camba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA other chlorinated organic compounds, dicamba does not persist (3,6-dichlorosalicylic acid). We recently described the in the soil because it is efficiently metabolized by a consortium biochemical characteristics of the three components of of soil bacteria under both aerobic and anaerobic conditions this enzyme (i.e. reductaseDIC, ferredoxinDIC, and oxy- (1–4). Studies with different soil types treated with dicamba genaseDIC) and classified the oxygenase component of have demonstrated that 3,6-dichlorosalicylic acid (DCSA),1 a dicamba O-demethylase as a member of the Rieske non- compound without herbicidal activity, is a major product of the heme iron family of oxygenases.
    [Show full text]
  • Flavin-Containing Monooxygenases: Mutations, Disease and Drug Response Phillips, IR; Shephard, EA
    Flavin-containing monooxygenases: mutations, disease and drug response Phillips, IR; Shephard, EA For additional information about this publication click this link. http://qmro.qmul.ac.uk/jspui/handle/123456789/1015 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] Flavin-containing monooxygenases: mutations, disease and drug response Ian R. Phillips1 and Elizabeth A. Shephard2 1School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK 2Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK Corresponding author: Shephard, E.A. ([email protected]). and, thus, contribute to drug development. This review Flavin-containing monooxygenases (FMOs) metabolize considers the role of FMOs and their genetic variants in numerous foreign chemicals, including drugs, pesticides disease and drug response. and dietary components and, thus, mediate interactions between humans and their chemical environment. We Mechanism and structure describe the mechanism of action of FMOs and insights For catalysis FMOs require flavin adenine dinucleotide gained from the structure of yeast FMO. We then (FAD) as a prosthetic group, NADPH as a cofactor and concentrate on the three FMOs (FMOs 1, 2 and 3) that are molecular oxygen as a cosubstrate [5,6]. In contrast to most important for metabolism of foreign chemicals in CYPs FMOs accept reducing equivalents directly from humans, focusing on the role of the FMOs and their genetic NADPH and, thus, do not require accessory proteins.
    [Show full text]
  • Identification of Novel CYP2E1 Inhibitor to Investigate Cellular and Exosomal CYP2E1-Mediated Toxicity
    University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 6-2019 Identification of Novel CYP2E1 Inhibitor to Investigate Cellular and Exosomal CYP2E1-Mediated Toxicity Mohammad Arifur Rahman University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Rahman, Mohammad Arifur (0000-0002-5589-0114), "Identification of Novel CYP2E1 Inhibitor to Investigate Cellular and Exosomal CYP2E1-Mediated Toxicity" (2019). Theses and Dissertations (ETD). Paper 482. http://dx.doi.org/10.21007/etd.cghs.2019.0474. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Identification of Novel CYP2E1 Inhibitor to Investigate Cellular and Exosomal CYP2E1-Mediated Toxicity Abstract Cytochrome P450 2E1 (CYP2E1)-mediated hepatic and extra-hepatic toxicity is of significant clinical importance. Diallyl sulfide (DAS) has been shown to prevent xenobiotics such as alcohol- (ALC/ETH), acetaminophen- (APAP) induced toxicity and disease (e.g. HIV-1) pathogenesis. DAS imparts its beneficial effect by inhibiting CYP2E1-mediated metabolism of xenobiotics, especially at high concentration. However, DAS also causes toxicity at relatively high dosages and with long exposure times. The objective of the first project was to find potent ASD analogs which can replace DAS as a research tool or as potential adjuvant therapy in CYP2E1-mediated pathologies.
    [Show full text]
  • Human Alkb Homologue 5 Is a Nuclear 2-Oxoglutarate Dependent Oxygenase and a Direct Target of Hypoxia- Inducible Factor 1A (HIF-1A)
    Human AlkB Homologue 5 Is a Nuclear 2-Oxoglutarate Dependent Oxygenase and a Direct Target of Hypoxia- Inducible Factor 1a (HIF-1a) Armin Thalhammer2, Zuzana Bencokova3., Rachel Poole3., Christoph Loenarz2., Julie Adam1, Linda O’Flaherty1, Johannes Scho¨ del1, David Mole1, Konstantinos Giaslakiotis4, Christopher J. Schofield2, Ester M. Hammond3, Peter J. Ratcliffe1, Patrick J. Pollard1* 1 Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, United Kingdom, 2 Chemistry Research Laboratory and The Oxford Centre for Integrative Systems Biology, Department of Chemistry, University of Oxford, Oxford, United Kingdom, 3 Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom, 4 Department of Cellular Pathology, John Radcliffe Hospital, Oxford, United Kingdom Abstract Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.
    [Show full text]
  • Card Uses a Minor Groove Wedge Mechanism to Stabilize the RNA
    1 CarD uses a minor groove wedge mechanism to stabilize the RNA 2 polymerase open promoter complex 3 4 Brian Bae1, James Chen1, Elizabeth Davis1, Katherine Leon1, Seth A. Darst1,*, 5 Elizabeth A. Campbell1,* 6 7 1The Rockefeller University, Laboratory for Molecular Biophysics, 1230 York Avenue, New York, 8 NY 10065, USA. 9 10 *Correspondence to: E-mail: [email protected], [email protected] 11 12 Present Address: Elizabeth Davis, The University of Minnesota School of Medicine, 13 420 Delaware St. SE, Minneapolis, MN 55455, USA; Katherine Leon, Department of 14 Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, GCIS 15 W219 Chicago, IL 60637, USA. 16 17 2 18 Abstract A key point to regulate gene expression is at transcription initiation, and 19 activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, 20 is found in many bacteria, including Thermus species, but absent in Escherichia coli. To 21 delineate the molecular mechanism of CarD, we determined crystal structures of 22 Thermus transcription initiation complexes containing CarD. The structures show CarD 23 interacts with the unique DNA topology presented by the upstream double- 24 stranded/single-stranded DNA junction of the transcription bubble. We confirm that our 25 structures correspond to functional activation complexes, and extend our understanding 26 of the role of a conserved CarD Trp residue that serves as a minor groove wedge, 27 preventing collapse of the transcription bubble to stabilize the transcription initiation 28 complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter 29 complexes, explaining the need for CarD.
    [Show full text]
  • Animal Evolution: Looking for the First Nervous System
    Dispatch R655 cellular or symbiotic functions, cannot genome reduction in the symbiont, 10. Nakabachi, A., Ishida, K., Hongoh, Y., Ohkuma, M., and Miyagishima, S. (2014). be lost without harmful or fatal results. HGT from various sources to the host Aphid gene of bacterial origin encodes a But these genes can be replaced, genome to maintain symbiont function, protein transported to an obligate and perhaps ever more easily as and now the targeting of protein endosymbiont. Curr. Biol. 24, R640–R641. 11. Moran, N.A., and Jarvik, T. (2010). Lateral the interaction networks of the products from host to symbiont has transfer of genes from fungi underlies endosymbiont reduce in complexity, even been found [4,10]. These make carotenoid production in aphids. Science 328, 624–627. thus reducing pressures on proteins clean separation of endosymbiont from 12. Jorgenson, M.A., Chen, Y., Yahashiri, A., to co-evolve. In a sense, when genes organelle more difficult to see, Popham, D.L., and Weiss, D.S. (2014). The are transferred from symbiont or prompting us not to look for the point bacterial septal ring protein RlpA is a lytic transglycosylase that contributes to rod organelle to the host nuclear genome when a symbiont ‘becomes’ an shape and daughter cell separation in and their proteins targeted back, this is organelle, but rather to ask, ‘Is there Pseudomonas aeruginosa. Mol. Microbiol. 93, 113–128. a compensatory change [4,9,14,17]. really anything so special about 13. Acuna, R., Padilla, B.E., Florez-Ramos, C.P., But other compensatory transfers that organelles?’ Rubio, J.D., Herrera, J.C., Benavides, P., affect a symbiont or organelle can also Lee, S.J., Yeats, T.H., Egan, A.N., Doyle, J.J., et al.
    [Show full text]