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1
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Sahtout N, Sanders DAR. Biophysical and biochemical characterization of the thioredoxin system from Colwellia psychrerythraea. Proteins 2024; 92:370-383. [PMID: 37909486 DOI: 10.1002/prot.26627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 09/24/2023] [Accepted: 10/17/2023] [Indexed: 11/03/2023]
Abstract
The thioredoxin system is a ubiquitous oxidoreductase system consisting of the enzyme thioredoxin reductase, the protein thioredoxin, and the cofactor nicotinamide adenine dinucleotide phosphate. The system has been comprehensively studied from many organisms, such as Escherichia coli; however, structural and functional analysis of this system from psychrophilic bacteria has not been as extensive. In this study, the thioredoxin system proteins of a psychrophilic bacterium, Colwellia psychrerythraea, were characterized using biophysical and biochemical techniques. Analysis of the complete genome sequence of the C. psychrerythraea thioredoxin system suggested the presence of a putative thioredoxin reductase and at least three thioredoxin. In this study, these identified putative thioredoxin system components were cloned, overexpressed, purified, and characterized. Our studies have indicated that the thioredoxin system proteins from E. coli were more stable than those from C. psychrerythraea. Consistent with these results, kinetic assays indicated that the thioredoxin reductase from E. coli had a higher optimal temperature than that from C. psychrerythraea.
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Affiliation(s)
- Naheda Sahtout
- Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
| | - David A R Sanders
- Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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2
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Wu S, Chen Y, Chen Z, Wei F, Zhou Q, Li P, Gu Q. Reactive oxygen species and gastric carcinogenesis: The complex interaction between Helicobacter pylori and host. Helicobacter 2023; 28:e13024. [PMID: 37798959 DOI: 10.1111/hel.13024] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Revised: 09/10/2023] [Accepted: 09/21/2023] [Indexed: 10/07/2023]
Abstract
Helicobacter pylori (H. pylori) is a highly successful human pathogen that colonizes stomach in around 50% of the global population. The colonization of bacterium induces an inflammatory response and a substantial rise in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mostly derived from host neutrophils and gastric epithelial cells, which play a crucial role in combating bacterial infections. However, H. pylori has developed various strategies to quench the deleterious effects of ROS, including the production of antioxidant enzymes, antioxidant proteins as well as blocking the generation of oxidants. The host's inability to eliminate H. pylori infection results in persistent ROS production. Notably, excessive ROS can disrupt the intracellular signal transduction and biological processes of the host, incurring chronic inflammation and cellular damage, such as DNA damage, lipid peroxidation, and protein oxidation. Markedly, the sustained inflammatory response and oxidative stress during H. pylori infection are major risk factor for gastric carcinogenesis. In this context, we summarize the literature on H. pylori infection-induced ROS production, the strategies used by H. pylori to counteract the host response, and subsequent host damage and gastric carcinogenesis.
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Affiliation(s)
- Shiying Wu
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
| | - Yongqiang Chen
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
| | - Ziqi Chen
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
| | - Fangtong Wei
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
| | - Qingqing Zhou
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
| | - Ping Li
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
| | - Qing Gu
- Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
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3
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Zheng C, Guo S, Tennant WG, Pradhan PK, Black KA, Dos Santos PC. The Thioredoxin System Reduces Protein Persulfide Intermediates Formed during the Synthesis of Thio-Cofactors in Bacillus subtilis. Biochemistry 2019; 58:1892-1904. [PMID: 30855939 DOI: 10.1021/acs.biochem.9b00045] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The biosynthesis of Fe-S clusters and other thio-cofactors requires the participation of redox agents. A shared feature in these pathways is the formation of transient protein persulfides, which are susceptible to reduction by artificial reducing agents commonly used in reactions in vitro. These agents modulate the reactivity and catalytic efficiency of biosynthetic reactions and, in some cases, skew the enzymes' kinetic behavior, bypassing sulfur acceptors known to be critical for the functionality of these pathways in vivo. Here, we provide kinetic evidence for the selective reactivity of the Bacillus subtilis Trx (thioredoxin) system toward protein-bound persulfide intermediates. Our results demonstrate that the redox flux of the Trx system modulates the rate of sulfide production in cysteine desulfurase assays. Likewise, the activity of the Trx system is dependent on the rate of persulfide formation, suggesting the occurrence of coupled reaction schemes between both enzymatic systems in vitro. Inactivation of TrxA (thioredoxin) or TrxR (thioredoxin reductase) impairs the activity of Fe-S enzymes in B. subtilis, indicating the involvement of the Trx system in Fe-S cluster metabolism. Surprisingly, biochemical characterization of TrxA reveals that this enzyme is able to coordinate Fe-S species, resulting in the loss of its reductase activity. The inactivation of TrxA through the coordination of a labile cluster, combined with its proposed role as a physiological reducing agent in sulfur transfer pathways, suggests a model for redox regulation. These findings provide a potential link between redox regulation and Fe-S metabolism.
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Affiliation(s)
- Chenkang Zheng
- Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27106 , United States
| | - Selina Guo
- Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27106 , United States
| | - William G Tennant
- Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27106 , United States
| | - Pradyumna K Pradhan
- Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27106 , United States.,Department of Chemistry and Biochemistry , The University of North Carolina at Greensboro , Greensboro , North Carolina 27412 , United States
| | - Katherine A Black
- Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27106 , United States.,Department of Medicine , Weill Cornell Medicine , New York , New York 10065 , United States
| | - Patricia C Dos Santos
- Department of Chemistry , Wake Forest University , Winston-Salem , North Carolina 27106 , United States
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4
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Nguyen K, DeSieno MA, Bae B, Johannes TW, Cobb RE, Zhao H, Nair SK. Characterization of the flavin monooxygenase involved in biosynthesis of the antimalarial FR-900098. Org Biomol Chem 2019; 17:1506-1518. [PMID: 30681110 DOI: 10.1039/c8ob02840k] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto 3-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal structure and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a similar overall structure with flavin-containing monooxygenases (FMOs). Notably, we also show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-binding domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.
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Affiliation(s)
- Kim Nguyen
- Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA
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5
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Bose M, Bhattacharyya S, Biswas R, Roychowdhury A, Bhattacharjee A, Ghosh AK, Das AK. Elucidation of the mechanism of disulfide exchange between staphylococcal thioredoxin2 and thioredoxin reductase2: A structural insight. Biochimie 2019; 160:1-13. [PMID: 30710560 DOI: 10.1016/j.biochi.2019.01.019] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2018] [Accepted: 01/28/2019] [Indexed: 11/20/2022]
Abstract
The redox homeostasis of cytoplasm is maintained by a series of disulfide exchange reactions mediated by proteins belonging to the thioredoxin superfamily. Thioredoxin and thioredoxin reductase, being the major members of the family, play a key role in oxidative stress response of Staphylococcus aureus. In this report, we have identified and characterised an active thioredoxin system of the mentioned pathogen. Crystal structure of thioredoxin2 (SaTrx2) in its reduced form reveals that it contains the conserved redox active WCXXC motif and a thioredoxin fold. Thioredoxin reductase2 (SaTR2) is a flavoprotein and consists of two Rossmann folds as the binding sites for FAD and NADPH. Crystal structure of the SaTR2 holoenzyme shows that the protein consists of two domains and the catalytic site comprises of an intramolecular disulfide bond formed between two sequentially distal cysteine residues. Biophysical and biochemical studies unveil that SaTrx2 and SaTR2 can physically interact in solution and in the course of sustaining the redox equilibrium, the latter reduces the former. Molecular docking has been performed to illustrate the interface formed between SaTrx2 and SaTR2 during the disulfide exchange reaction.
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Affiliation(s)
- Madhuparna Bose
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Sudipta Bhattacharyya
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Rupam Biswas
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Amlan Roychowdhury
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Atanu Bhattacharjee
- Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, 793022, India
| | - Ananta Kumar Ghosh
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India
| | - Amit Kumar Das
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
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6
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Buey RM, Schmitz RA, Buchanan BB, Balsera M. Crystal Structure of the Apo-Form of NADPH-Dependent Thioredoxin Reductase from a Methane-Producing Archaeon. Antioxidants (Basel) 2018; 7:E166. [PMID: 30453601 PMCID: PMC6262447 DOI: 10.3390/antiox7110166] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 11/12/2018] [Accepted: 11/14/2018] [Indexed: 12/20/2022] Open
Abstract
The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.
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Affiliation(s)
- Rubén M Buey
- Metabolic Engineering Group. Dpto. Microbiología y Genética. Universidad de Salamanca, 37007 Salamanca, Spain.
| | - Ruth A Schmitz
- Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany.
| | - Bob B Buchanan
- Department of Plant & Microbial Biology, University of California, 94720 Berkeley CA, USA.
| | - Monica Balsera
- Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), 37008 Salamanca, Spain.
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Valette O, Tran TTT, Cavazza C, Caudeville E, Brasseur G, Dolla A, Talla E, Pieulle L. Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris. Front Microbiol 2017; 8:1855. [PMID: 29033913 PMCID: PMC5627308 DOI: 10.3389/fmicb.2017.01855] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2017] [Accepted: 09/11/2017] [Indexed: 11/19/2022] Open
Abstract
Thioredoxin reductase (TR) regulates the intracellular redox environment by reducing thioredoxin (Trx). In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH), there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi). The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(P)H-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2′-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time.
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Affiliation(s)
| | - Tam T T Tran
- Aix-Marseille Univ, CNRS, LCB, Marseille, France
| | - Christine Cavazza
- Laboratoire de Chimie et Biologie des Métaux, Université Grenoble Alpes, Grenoble, France.,UMR 5249, Laboratoire de Chimie et Biologie des Métaux, Centre National de la Recherche Scientifique, Grenoble, France.,DRF/BIG/CBM, CEA-Grenoble, Grenoble, France
| | | | | | - Alain Dolla
- Aix-Marseille Univ, CNRS, LCB, Marseille, France
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8
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Brogi S, Fiorillo A, Chemi G, Butini S, Lalle M, Ilari A, Gemma S, Campiani G. Structural characterization of Giardia duodenalis thioredoxin reductase (gTrxR) and computational analysis of its interaction with NBDHEX. Eur J Med Chem 2017; 135:479-490. [PMID: 28477573 DOI: 10.1016/j.ejmech.2017.04.057] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2017] [Revised: 04/18/2017] [Accepted: 04/21/2017] [Indexed: 12/22/2022]
Abstract
Giardia duodenalis is a microaerophilic parasite that colonizes the upper portions of the small intestine of humans. Giardia infection is a major contributor to diarrheal disease worldwide. Nitroheterocycles (e.g. metronidazole) or benzimidazoles (e.g. albendazole) are the most commonly used therapeutic agents. Unfortunately, their efficacy is reduced by low compliance or resistance phenomena. We recently discovered that the antitumoral drug 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is active against G. duodenalis trophozoites and its mode of action is linked to inhibition of thioredoxin reductase (gTrxR), a key component of Giardia redox system: gTrxR provides efficient defenses against reactive oxygen species (ROS), it is a target of 5-nitroimidazoles antiparasitic drugs and also contributes to their metabolism. However, the exact mechanism responsible for the gTrxR inhibition mediated by this chemical class of antigiardial compounds is yet to be defined. The definition of the structural determinants of activity against gTrxR could be important for the identification of novel drugs endowed with an innovative mode of action. With this aim, we solved the crystal structure of gTrxR and we analyzed in silico the binding mode of NBDHEX. The data presented herein could guide the development of NBDHEX derivatives tailored for selective inhibition of gTrxR as antigiardial agents.
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Affiliation(s)
- Simone Brogi
- European Research Centre for Drug Discovery and Development (NatSynDrugs), Department of Biotechnology, Chemistry, and Pharmacy, Università di Siena via Aldo Moro 2, 53100 Siena, Italy
| | - Annarita Fiorillo
- CNR (Consiglio Nazionale delle Ricerche) - Istituto di Biologia e Patologia Molecolari (IBPM), c/o Dipartimento di Scienze Biochimiche P.le Aldo Moro 5, 00185, Roma, Italy
| | - Giulia Chemi
- European Research Centre for Drug Discovery and Development (NatSynDrugs), Department of Biotechnology, Chemistry, and Pharmacy, Università di Siena via Aldo Moro 2, 53100 Siena, Italy
| | - Stefania Butini
- European Research Centre for Drug Discovery and Development (NatSynDrugs), Department of Biotechnology, Chemistry, and Pharmacy, Università di Siena via Aldo Moro 2, 53100 Siena, Italy
| | - Marco Lalle
- Istituto Superiore di Sanità, Department of Infectious Diseases, viale Regina Elena 299, Rome, Italy.
| | - Andrea Ilari
- CNR (Consiglio Nazionale delle Ricerche) - Istituto di Biologia e Patologia Molecolari (IBPM), c/o Dipartimento di Scienze Biochimiche P.le Aldo Moro 5, 00185, Roma, Italy.
| | - Sandra Gemma
- European Research Centre for Drug Discovery and Development (NatSynDrugs), Department of Biotechnology, Chemistry, and Pharmacy, Università di Siena via Aldo Moro 2, 53100 Siena, Italy.
| | - Giuseppe Campiani
- European Research Centre for Drug Discovery and Development (NatSynDrugs), Department of Biotechnology, Chemistry, and Pharmacy, Università di Siena via Aldo Moro 2, 53100 Siena, Italy
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9
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Owings JP, McNair NN, Mui YF, Gustafsson TN, Holmgren A, Contel M, Goldberg JB, Mead JR. Auranofin and N-heterocyclic carbene gold-analogs are potent inhibitors of the bacteria Helicobacter pylori. FEMS Microbiol Lett 2016; 363:fnw148. [PMID: 27279627 DOI: 10.1093/femsle/fnw148] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/31/2016] [Indexed: 01/20/2023] Open
Abstract
Auranofin is an FDA-approved gold-containing compound used for the treatment of rheumatoid arthritis. Recent reports of antimicrobial activity against protozoa and bacteria indicate that auranofin targets the reductive enzyme thioredoxin reductase (TrxR). We evaluated auranofin as well as five auranofin analogs containing N-heterocyclic carbenes (instead of the triethylphosphane present in auranofin) and five gold-carbene controls for their ability to inhibit or kill Helicobacter pylori in vitro Auranofin completely inhibited bacterial growth at 1.2 μM. Purified H. pylori TrxR was inhibited by auranofin in a cell-free assay (IC50 ∼88 nM). The most active gold(I)-N-heterocyclic carbene compounds exhibited MICs comparable to auranofin against H. pylori (2 μM), while also exhibiting lower toxicities for human embryonic kidney cells (HEK-293T cells). Median toxic concentrations (TC50) were 13-20-fold higher compared to auranofin indicating that they were less cytotoxic. The N-heterocyclic carbene analogs maybe well tolerated, but further evaluation is needed in vivo Finally, auranofin was synergistic with the antibiotic amoxicillin, suggesting that targeting both the reductive enzyme TrxR and cell wall synthesis may be effective against H. pylori infections.
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Affiliation(s)
- Joshua P Owings
- Department of Pediatrics, 1760 Haygood, Emory University, Atlanta, GA 30022, USA
| | - Nina N McNair
- Department of Pediatrics, 1760 Haygood, Emory University, Atlanta, GA 30022, USA Department of Medical Research, Atlanta VA Medical Center, 1670 Clairmont Rd, Decatur, GA 30033, USA
| | - Yiu Fung Mui
- Department of Chemistry, Brooklyn College and The Graduate Center, The City University of New York, Brooklyn, NY 11210, USA Chemistry and Biology PhD Programs, The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, NY 10016, USA
| | - Tomas N Gustafsson
- Department of Medical Biochemistry, and Biophysics, Karolinska Institute, SE 171 77 Stockholm, Sweden
| | - Arne Holmgren
- Department of Medical Biochemistry, and Biophysics, Karolinska Institute, SE 171 77 Stockholm, Sweden
| | - Maria Contel
- Department of Chemistry, Brooklyn College and The Graduate Center, The City University of New York, Brooklyn, NY 11210, USA Chemistry and Biology PhD Programs, The Graduate Center, The City University of New York, 365 Fifth Avenue, New York, NY 10016, USA
| | - Joanna B Goldberg
- Department of Pediatrics, 1760 Haygood, Emory University, Atlanta, GA 30022, USA
| | - Jan R Mead
- Department of Pediatrics, 1760 Haygood, Emory University, Atlanta, GA 30022, USA Department of Medical Research, Atlanta VA Medical Center, 1670 Clairmont Rd, Decatur, GA 30033, USA
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10
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Gustafsson TN, Osman H, Werngren J, Hoffner S, Engman L, Holmgren A. Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis. Biochim Biophys Acta Gen Subj 2016; 1860:1265-71. [PMID: 26971857 DOI: 10.1016/j.bbagen.2016.03.013] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Revised: 02/25/2016] [Accepted: 03/07/2016] [Indexed: 12/15/2022]
Abstract
BACKGROUND Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. METHODS We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. RESULTS The most potent compounds in the series gave IC(50) values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 μM (0.12 μg/ml) for B. subtilis, 1.5 μM (0.64 μg/ml) for S. aureus, 2 μM (0.86 μg/ml) for B. cereus and 10 μg/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. CONCLUSIONS These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. GENERAL SIGNIFICANCE We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens.
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Affiliation(s)
- Tomas N Gustafsson
- Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institutet, Stockholm, Sweden; Department of Clinical Microbiology, Clinical Bacteriology, Sunderby Research Unit, Umeå University, Umeå, Sweden.
| | - Harer Osman
- Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institutet, Stockholm, Sweden
| | - Jim Werngren
- Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden
| | - Sven Hoffner
- Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden
| | - Lars Engman
- Department of Chemistry, BMC, Uppsala University, Uppsala, Sweden
| | - Arne Holmgren
- Department of Medical Biochemistry and Biophysics, Division of Biochemistry, Karolinska Institutet, Stockholm, Sweden.
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11
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Golden E, Karton A, Vrielink A. High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization. ACTA ACUST UNITED AC 2014; 70:3155-66. [PMID: 25478834 DOI: 10.1107/s139900471402286x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2014] [Accepted: 10/17/2014] [Indexed: 01/09/2023]
Abstract
Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features.
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Affiliation(s)
- Emily Golden
- School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia
| | - Amir Karton
- School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia
| | - Alice Vrielink
- School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia
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12
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Liu R, Wang Y, Yuan Q, An D, Li J, Gao X. The Au clusters induce tumor cell apoptosis via specifically targeting thioredoxin reductase 1 (TrxR1) and suppressing its activity. Chem Commun (Camb) 2014; 50:10687-90. [DOI: 10.1039/c4cc03320e] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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13
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The structural basis of an NADP⁺-independent dithiol oxidase in FK228 biosynthesis. Sci Rep 2014; 4:4145. [PMID: 24553401 PMCID: PMC4894451 DOI: 10.1038/srep04145] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Accepted: 02/05/2014] [Indexed: 01/06/2023] Open
Abstract
The disulfide bond is unusual in natural products and critical for thermal stability, cell permeability and bioactivity. DepH from Chromobacterium violaceum No. 968 is an FAD-dependent enzyme responsible for catalyzing the disulfide bond formation of FK228, an anticancer prodrug approved for the treatment of cutaneous T-cell lymphoma. Here we report the crystal structures of DepH and DepH complexed with a substrate analogue S,S'-dimethyl FK228 at 1.82 Å and 2.00 Å, respectively. Structural and biochemical analyses revealed that DepH, in contrast to the well characterized low molecular weight thioredoxin reductases (LMW TrxRs), is an NADP+-independent dithiol oxidase. DepH not only lacks a conserved GGGDXAXE motif necessary for NADP+ binding in the canonical LMW TrxRs, but also contains a 11-residue sequence which physically impedes the binding of NADP+. These observations explain the difference between NADP+-independent small molecule dithiol oxidases and NADP+-dependent thioredoxin reductases and provide insights for understanding the catalytic mechanism of dithiol oxidases involved in natural product biosynthesis.
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14
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Zanotti G, Cendron L. Structural and functional aspects of the Helicobacter pylori secretome. World J Gastroenterol 2014; 20:1402-1423. [PMID: 24587618 PMCID: PMC3925851 DOI: 10.3748/wjg.v20.i6.1402] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Accepted: 01/06/2014] [Indexed: 02/06/2023] Open
Abstract
Proteins secreted by Helicobacter pylori (H. pylori), an important human pathogen responsible for severe gastric diseases, are reviewed from the point of view of their biochemical characterization, both functional and structural. Despite the vast amount of experimental data available on the proteins secreted by this bacterium, the precise size of the secretome remains unknown. In this review, we consider as secreted both proteins that contain a secretion signal for the periplasm and proteins that have been detected in the external medium in in vitro experiments. In this way, H. pylori’s secretome appears to be composed of slightly more than 160 proteins, but this number must be considered very cautiously, not only because the definition of secretome itself is ambiguous but also because the included proteins were observed as secreted in in vitro experiments that were not representative of the environmental situation in vivo. The proteins that appear to be secreted can be grouped into different classes: enzymes (48 proteins), outer membrane proteins (43), components of flagella (11), members of the cytotoxic-associated genes pathogenicity island or other toxins (8 and 5, respectively), binding and transport proteins (9), and others (11). A final group, which includes 28 members, is represented by hypothetical uncharacterized proteins. Despite the large amount of data accumulated on the H. pylori secretome, a considerable amount of work remains to reach a full comprehension of the system at the molecular level.
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15
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Abstract
The thioredoxin (Trx) system, which is composed of NADPH, thioredoxin reductase (TrxR), and thioredoxin, is a key antioxidant system in defense against oxidative stress through its disulfide reductase activity regulating protein dithiol/disulfide balance. The Trx system provides the electrons to thiol-dependent peroxidases (peroxiredoxins) to remove reactive oxygen and nitrogen species with a fast reaction rate. Trx antioxidant functions are also shown by involvement in DNA and protein repair by reducing ribonucleotide reductase, methionine sulfoxide reductases, and regulating the activity of many redox-sensitive transcription factors. Moreover, Trx systems play critical roles in the immune response, virus infection, and cell death via interaction with thioredoxin-interacting protein. In mammalian cells, the cytosolic and mitochondrial Trx systems, in which TrxRs are high molecular weight selenoenzymes, together with the glutathione-glutaredoxin (Grx) system (NADPH, glutathione reductase, GSH, and Grx) control the cellular redox environment. Recently mammalian thioredoxin and glutathione systems have been found to be able to provide the electrons crossly and to serve as a backup system for each other. In contrast, bacteria TrxRs are low molecular weight enzymes with a structure and reaction mechanism distinct from mammalian TrxR. Many bacterial species possess specific thiol-dependent antioxidant systems, and the significance of the Trx system in the defense against oxidative stress is different. Particularly, the absence of a GSH-Grx system in some pathogenic bacteria such as Helicobacter pylori, Mycobacterium tuberculosis, and Staphylococcus aureus makes the bacterial Trx system essential for survival under oxidative stress. This provides an opportunity to kill these bacteria by targeting the TrxR-Trx system.
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Affiliation(s)
- Jun Lu
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
| | - Arne Holmgren
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
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16
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Lu J, Vodnala SK, Gustavsson AL, Gustafsson TN, Sjöberg B, Johansson HA, Kumar S, Tjernberg A, Engman L, Rottenberg ME, Holmgren A. Ebsulfur is a benzisothiazolone cytocidal inhibitor targeting the trypanothione reductase of Trypanosoma brucei. J Biol Chem 2013; 288:27456-27468. [PMID: 23900839 DOI: 10.1074/jbc.m113.495101] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.
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Affiliation(s)
- Jun Lu
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics
| | | | - Anna-Lena Gustavsson
- Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden
| | - Tomas N Gustafsson
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics
| | - Birger Sjöberg
- Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine, Karolinska Institutet, SE-17177 Stockholm, Sweden
| | - Henrik A Johansson
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics; Department of Chemistry-BMC, Uppsala University, SE-751 23 Uppsala, Sweden
| | | | | | - Lars Engman
- Department of Chemistry-BMC, Uppsala University, SE-751 23 Uppsala, Sweden
| | | | - Arne Holmgren
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics.
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17
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Yamada M, Tamada T, Takeda K, Matsumoto F, Ohno H, Kosugi M, Takaba K, Shoyama Y, Kimura S, Kuroki R, Miki K. Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer. J Mol Biol 2013; 425:4295-306. [PMID: 23831226 DOI: 10.1016/j.jmb.2013.06.010] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2013] [Revised: 05/18/2013] [Accepted: 06/12/2013] [Indexed: 10/26/2022]
Abstract
NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD(+). Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.
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Affiliation(s)
- Mitsugu Yamada
- Molecular Biology Research Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-shirane, Tokai, Ibaraki 319-1195, Japan
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18
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Koch O, Jäger T, Heller K, Khandavalli PC, Pretzel J, Becker K, Flohé L, Selzer PM. Identification of M. tuberculosis Thioredoxin Reductase Inhibitors Based on High-Throughput Docking Using Constraints. J Med Chem 2013; 56:4849-59. [DOI: 10.1021/jm3015734] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- Oliver Koch
- MSD Animal Health Innovation GmbH, Schwabenheim, Germany
- MOLISA GmbH, Magdeburg,
Germany
| | - Timo Jäger
- MOLISA GmbH, Magdeburg,
Germany
- Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany
| | | | | | - Jette Pretzel
- Biochemie und Molekularbiologie, Justus-Liebig-Universität, Giessen, Germany
| | - Katja Becker
- Biochemie und Molekularbiologie, Justus-Liebig-Universität, Giessen, Germany
| | | | - Paul M. Selzer
- MSD Animal Health Innovation GmbH, Schwabenheim, Germany
- Interfakultäres
Institut für
Biochemie, Universität Tübingen, Tübingen, Germany
- Wellcome Trust Centre for Molecular
Parasitology and Division of Infection and Immunity, University of Glasgow, Glasgow, U.K
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19
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Lu J, Vlamis‐Gardikas A, Kandasamy K, Zhao R, Gustafsson TN, Engstrand L, Hoffner S, Engman L, Holmgren A. Inhibition of bacterial thioredoxin reductase: an antibiotic mechanism targeting bacteria lacking glutathione. FASEB J 2012; 27:1394-403. [DOI: 10.1096/fj.12-223305] [Citation(s) in RCA: 113] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Jun Lu
- Division of BiochemistryDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
| | - Alexios Vlamis‐Gardikas
- Division of BiochemistryDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
| | - Karuppasamy Kandasamy
- Division of BiochemistryDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
| | - Rong Zhao
- Division of BiochemistryDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
| | - Tomas N. Gustafsson
- Division of BiochemistryDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
| | - Lars Engstrand
- Microbiology and Tumor Biology CenterKarolinska InstitutetStockholmSweden
| | - Sven Hoffner
- Microbiology and Tumor Biology CenterKarolinska InstitutetStockholmSweden
- World Health Organization Supranational Tuberculosis Reference LaboratoryDepartment for PreparednessSwedish Institute for Communicable Disease ControlSolnaSweden
| | - Lars Engman
- Department of Biochemistry and Organic ChemistryUppsala UniversityUppsalaSweden
| | - Arne Holmgren
- Division of BiochemistryDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
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20
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Gustafsson TN, Sahlin M, Lu J, Sjöberg BM, Holmgren A. Bacillus anthracis thioredoxin systems, characterization and role as electron donors for ribonucleotide reductase. J Biol Chem 2012; 287:39686-97. [PMID: 23012357 DOI: 10.1074/jbc.m112.413427] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.
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Affiliation(s)
- Tomas N Gustafsson
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet SE-17177 Stockholm, Sweden
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21
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Zanotti G, Cendron L. Functional and structural aspects of Helicobacter pylori acidic stress response factors. IUBMB Life 2011; 62:715-23. [PMID: 20960531 DOI: 10.1002/iub.382] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Helicobacter pylori is a striking example of adaptation of a bacterium to a very peculiar niche, the human stomach. Despite being a neutralophile, a sophisticated control of gene expression allows it to live and to proliferate in an environment that cycles from nearly neutral to very acidic. Despite the numerous studies performed on the mechanisms of acid adaptation, the physiological function of a large part of the genes products that are up-regulated or down-regulated is often not clear, in particular in the context of the response of the bacterium to an acidic stress. In this review, we discuss the molecular and functional aspects of some of the proteins that are commonly found overexpressed during the acid stress.
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Affiliation(s)
- Giuseppe Zanotti
- Department of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.
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22
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Obiero J, Sanders DAR. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis. Protein Sci 2011; 20:1021-9. [PMID: 21465613 DOI: 10.1002/pro.635] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2011] [Revised: 03/22/2011] [Accepted: 03/22/2011] [Indexed: 11/06/2022]
Abstract
In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.
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Affiliation(s)
- Josiah Obiero
- Department of Chemistry, University of Saskatchewan, Saskatoon, SK S7N5C9, Canada
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23
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Negri A, Rodríguez-Larrea D, Marco E, Jiménez-Ruiz A, Sánchez-Ruiz JM, Gago F. Protein-protein interactions at an enzyme-substrate interface: characterization of transient reaction intermediates throughout a full catalytic cycle of Escherichia coli thioredoxin reductase. Proteins 2010; 78:36-51. [PMID: 19585660 DOI: 10.1002/prot.22490] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X-ray crystallographic structures of dimeric wild-type TrxR in the flavin-oxidizing conformation and a C135S TrxR mutant enzyme in a flavin-reducing conformation "trapped" by a cross-link between Cys138 of TrxR and Cys32 of C35S mutant thioredoxin (Trx). The transition between these two extreme states, which is shown to be reproduced in a normal mode analysis, as well as natural cofactor binding and dissociation, were simulated for the wild-type species using unrestrained and targeted molecular dynamics following docking of oxidized Trx to reduced TrxR. The whole set of simulations provides a comprehensive structural framework for understanding the mechanism of disulfide reduction in atomic detail and identifying the most likely intermediates that facilitate entry of NADPH and exit of NADP(+). The crucial role assigned to Arg73 and Lys36 of Trx in substrate binding and complex stabilization was ascertained when R73G, R73D, and K36A site-directed mutants of Trx were shown to be impaired to different extents in their ability to be reduced by TrxR. On the basis of previous findings and the results reported herein, E. coli TrxR appears as a beautifully engineered molecular machine that is capable of synchronizing cofactor capture and ejection with substrate binding and redox activity through an interdomain twisting motion.
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Affiliation(s)
- Ana Negri
- Departmento de Farmacología, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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24
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Denschlag R, Schreier WJ, Rieff B, Schrader TE, Koller FO, Moroder L, Zinth W, Tavan P. Relaxation time prediction for a light switchable peptide by molecular dynamics. Phys Chem Chem Phys 2010; 12:6204-18. [DOI: 10.1039/b921803c] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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25
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Duckworth MJ, Okoli AS, Mendz GL. Novel Helicobacter pylori therapeutic targets: the unusual suspects. Expert Rev Anti Infect Ther 2009; 7:835-67. [PMID: 19735225 DOI: 10.1586/eri.09.61] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Understanding the current status of the discovery and development of anti-Helicobacter therapies requires an overview of the searches for therapeutic targets performed to date. A summary is given of the very substantial body of work conducted in the quest to find Helicobacter pylori genes that could be suitable candidates for therapeutic intervention. The products of most of these genes perform metabolic functions, and others have roles in growth, cell motility and colonization. The genes identified as potential targets have been organized into three categories according to their degree of characterization. A short description and evaluation is provided of the main candidates in each category. Investigations of potential therapeutic targets have generated a wealth of information about the physiology and genetics of H. pylori, and its interactions with the host, but have yielded little by way of new therapies.
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Affiliation(s)
- Megan J Duckworth
- School of Medicine, Sydney, The University of Notre Dame Australia, 160 Oxford Street, Darlinghurst, NSW 2010, Australia.
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26
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Abstract
This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-A resolution. The overall structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K(m) (5.7 muM) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K(m), 44.4 muM). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.
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27
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Kirkensgaard KG, Hägglund P, Finnie C, Svensson B, Henriksen A. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism. ACTA CRYSTALLOGRAPHICA. SECTION D, BIOLOGICAL CRYSTALLOGRAPHY 2009; 65:932-41. [PMID: 19690371 PMCID: PMC2733882 DOI: 10.1107/s0907444909021817] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/04/2009] [Accepted: 06/09/2009] [Indexed: 12/24/2022]
Abstract
Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 A resolution and refined to an R(cryst) of 19.0% and an R(free) of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25 degrees and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR-Trx interactions mediate the FO to FR transformation.
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Affiliation(s)
- Kristine G. Kirkensgaard
- Carlsberg Laboratory, Denmark
- Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark, Denmark
| | - Per Hägglund
- Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark, Denmark
| | - Christine Finnie
- Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark, Denmark
| | - Birte Svensson
- Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark, Denmark
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28
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Lu J, Berndt C, Holmgren A. Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase. Biochim Biophys Acta Gen Subj 2009; 1790:1513-9. [PMID: 19406206 DOI: 10.1016/j.bbagen.2009.04.013] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2009] [Revised: 04/21/2009] [Accepted: 04/22/2009] [Indexed: 01/24/2023]
Abstract
The mammalian thioredoxin reductases (TrxR) are selenoproteins with a catalytic selenocysteine residue which in the oxidized enzyme forms a selenenylsulfide and in the reduced enzyme is present as a selenolthiol. Selenium compounds such as selenite, selenodiglutathione and selenocystine are substrates for the enzyme with low K(m)-values and the enzyme is implicated in reductive assimilation of selenium by generating selenide for selenoprotein synthesis. Redox cycling of reduced metabolites of these selenium compounds including selenide with oxygen via TrxR and reduced thioredoxin (Trx) will oxidize NADPH and produce reactive oxygen species inducing cell death at high concentrations explaining selenite toxicity. There is no free pool of selenocysteine since this would be toxic in an oxygen environment by redox cycling via thioredoxin systems. The importance of selenium compounds and TrxR in cancer and cardiovascular diseases both for prevention and treatment is discussed. A selenazol drug like ebselen is a direct substrate for mammalian TrxR and dithiol Trx and ebselen selenol is readily reoxidized by hydrogen peroxide and lipid hydroperoxides, acting as an anti-oxidant and anti-inflammatory drug.
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Affiliation(s)
- Jun Lu
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77 Stockholm, Sweden
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Zhang Z, Bao R, Zhang Y, Yu J, Zhou CZ, Chen Y. Crystal structure of Saccharomyces cerevisiae cytoplasmic thioredoxin reductase Trr1 reveals the structural basis for species-specific recognition of thioredoxin. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2009; 1794:124-8. [DOI: 10.1016/j.bbapap.2008.09.011] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/10/2008] [Revised: 08/15/2008] [Accepted: 09/16/2008] [Indexed: 11/25/2022]
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