DOCSLIB.ORG

Interactive Control of Carbon Assimilation, Redox Balance, CBB

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Interactive Control of Carbon Assimilation, Redox Balance, CBB Interactive Control of Carbon Assimilation, Redox Balance, CBB Expression, Nitrogenase Complex Biosynthesis, Hydrogen Production, and Sulfur Metabolism in RubisCO Compromised Mutant Strains of Nonsulfur Purple Bacteria Dissertation Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Rick A. Laguna, B.S. Graduate Program in Microbiology The Ohio State University 2010 Dissertation Committee F.R. Tabita, Advisor B. Alber C.J. Daniels J.A. Krzycki Copyright by Rick A. Laguna 2010 Abstract The continuation of redox homeostasis is required for bacterial metabolism and cellular integrity. In order to maintain redox balance, multifaceted and integrated regulatory networks are required to respond to variable inter- and intracellular environments. The ability of nonsulfur purple (NSP) photosynthetic bacteria to maintain redox poise during photoheterotrophic growth is remarkable. Crosstalk and coordinated regulation between various systems (DMSO reduction, CO2- and N2-fixation) are employed to maintain redox poise. The use of each system is influenced either by the availability of the preferred terminal electron acceptor or the presence or absence of one system. For example, in Rhodobacter sphaeroides, excess reductant generated during photoheterotrophic growth is consumed by the reduction of metabolically produced CO2 via ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) through the Calvin- Benson-Bassham (CBB) reductive pentose phosphate CO2 fixation pathway. However, when the primary electron sink, the reduction of CO2 is void due to the presence of a non-functional CBB cycle, the use of alternative redox sinks must be employed. For example, derepression of nitrogenase complex synthesis under normal repressive conditions, allows for cellular redox balance through reduction of protons to H2. To further understand cellular processes that are involved in synthesis and consumption of cellular reductant, which can maximize the production of bio-fuels in NSP bacteria, in this dissertation we characterize and probe the role of the CBB ii cycle in Rhodopseudomonas palustris and Rb. sphaeroides. In addition, we confirm that synthesis of nitrogenase complex and the use of this protein complex to produce hydrogen gas is intimately tied to the regulation of the CBB pathway. Moreover, we show that a mutation in the nifA gene of Rps. palustris RubisCO deletion strain CGA2044 allowed for expression and synthesis of the nitrogenase complex. On the other hand, mutations in both the nifA and glnA genes appeared to allow for expression and synthesis of the nitrogenase complex in Rb. sphaeroides RubisCO deletion strain 16PHC. Further, it was found that Rps. palustris RubisCO deletion strain CGA2044 produced the most hydrogen as compared to other RubisCO deletion strains of NSP photosynthetic bacteria and inactivation of the CBB cycle increased hydrogen production. Rb. sphaeroides RubisCO deletion strain 17 was also isolated and it was shown that expression of the CBB system was down-regulated in this strain as compared to wild-type. The cbbR gene in Rb. sphaeroides RubisCO deletion strains 16, 17, and 16PHC was up-regulated as compared to wild-type. Finally, in Rb. sphaeroides RubisCO deletion strain 16PHG, sulfate reduction, serine-, and cysteine-biosynthesis appeared to play roles in cellular redox balance within this strain, with sulfite reductase, phosphoglycerate dehydrogenase, and cysteine synthase up-regulated as compared to wild-type. In strain 16PHG, the production of hydrogen sulfide was derived from reduction of sulfate and the ultimate alternative redox sink appeared to be down-stream of cysteine metabolism. iii Dedication In memory of Dr. Thomas M. Wahlund, a great friend and mentor. iv Acknowledgements I would like to thank Dr. Tabita, my committee members, and fellow lab workers for guidance and input during my graduate studies. I would also like to thank my family, especially my mom and dad, for their continued support. Last, but not least, I would like to thank my wonderful wife, Kelly. I am extremely grateful for your continued support, encouragement, and understanding. v Vita 2001.………………………………………..B.S., Biological Sciences, California State University San Marcos, San Marcos, CA 2001 - present……………………………..Graduate Research and Teaching Associate, Department of Microbiology The Ohio State University, Columbus, OH Publications Laguna R, Tabita FR, Alber BE. Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin-Benson-Bassham reductive pentose phosphate pathway in Rhodobacter sphaeroides and Rhodopseudomonas palustris. In Press. 2010. Rizk ML, Laguna R, Smith KM, Tabita FR, and Liao JC. Redox homeostasis phenotypes in RubisCO-deficient Rhodobacter sphaeroides via ensemble modeling. In Press. 2010. Laguna R, Joshi GS, Dangel AW, Luther AK, Tabita FR. Integrative control of carbon, nitrogen, hydrogen, and sulfur metabolism: the central role of the Calvin-Benson- Bassham cycle. Adv Exp Med Biol. 2010;675:265-71. Zianni M, Tessanne K, Merighi M, Laguna R, Tabita FR. Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument. J Biomol Tech. 2006 Apr;17(2):103-13. Laguna R, Romo J, Read BA, Wahlund TM. Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi. Appl Environ Microbiol. 2001 Sep;67(9):3824-31. Field of Study Major Field: Microbiology vi Table of Contents ABSTRACT ............................................................................................................................................. II ACKNOWLEDGEMENTS ....................................................................................................................V VITA ..........................................................................................................................................................VI LIST OF TABLES..............................................................................................................................VIII LIST OF FIGURES............................................................................................................................... IX LIST OF ABBREVATIONS................................................................................................................ XI INTRODUCTION.....................................................................................................................................1 CHAPTER 1. CELLULAR REDOX BALANCE AND CARBON ASSIMILATION IN RHODOBACTER SPHAEROIDES AND RHODOPSEUDOMONAS PALUSTRIS. ..........4 INTRODUCTION........................................................................................................................................................4 MATERIALS AND METHODS................................................................................................................................6 DISCUSSION ..........................................................................................................................................................17 CHAPTER 2. NIFA AND OTHER MUTATIONS AND THEIR INFLUENCE ON HYDROGEN PRODUCTION IN RUBISCO COMPROMISED MUTANT STRAINS OF NONSULFUR PURPLE BACTERIA. ............................................................................................ 18 INTRODUCTION.....................................................................................................................................................18 MATERIALS AND METHODS.............................................................................................................................23 RESULTS.................................................................................................................................................................28 DISCUSSION ..........................................................................................................................................................41 CHAPTER 3. TRANSCRIPTOME ANALYSIS OF RHODOBACTER SPHAEROIDES RUBSICO COMPROMISED MUTANT STRAINS ..................................................................... 45 INTRODUCTION.....................................................................................................................................................45 RESULTS.................................................................................................................................................................50 CONCLUSION ........................................................................................................................................................61 CHAPTER 4. PRELIMINARY CHARACTERIZATION OF AN ALTERNATIVE REDOX SINK IN RHODOBACTER SPHAEROIDES RUBISCO DELETION MUTANT STRAIN 16PHG..................................................................................................................................................... 64 INTRODUCTION.....................................................................................................................................................64 RESULTS.................................................................................................................................................................70 DISCUSSION ..........................................................................................................................................................74
Load more

Recommended publications
  • Enzymatic Encoding Methods for Efficient Synthesis Of
    (19) TZZ__T (11) EP 1 957 644 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/10 (2006.01) C12Q 1/68 (2006.01) 01.12.2010 Bulletin 2010/48 C40B 40/06 (2006.01) C40B 50/06 (2006.01) (21) Application number: 06818144.5 (86) International application number: PCT/DK2006/000685 (22) Date of filing: 01.12.2006 (87) International publication number: WO 2007/062664 (07.06.2007 Gazette 2007/23) (54) ENZYMATIC ENCODING METHODS FOR EFFICIENT SYNTHESIS OF LARGE LIBRARIES ENZYMVERMITTELNDE KODIERUNGSMETHODEN FÜR EINE EFFIZIENTE SYNTHESE VON GROSSEN BIBLIOTHEKEN PROCEDES DE CODAGE ENZYMATIQUE DESTINES A LA SYNTHESE EFFICACE DE BIBLIOTHEQUES IMPORTANTES (84) Designated Contracting States: • GOLDBECH, Anne AT BE BG CH CY CZ DE DK EE ES FI FR GB GR DK-2200 Copenhagen N (DK) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • DE LEON, Daen SK TR DK-2300 Copenhagen S (DK) Designated Extension States: • KALDOR, Ditte Kievsmose AL BA HR MK RS DK-2880 Bagsvaerd (DK) • SLØK, Frank Abilgaard (30) Priority: 01.12.2005 DK 200501704 DK-3450 Allerød (DK) 02.12.2005 US 741490 P • HUSEMOEN, Birgitte Nystrup DK-2500 Valby (DK) (43) Date of publication of application: • DOLBERG, Johannes 20.08.2008 Bulletin 2008/34 DK-1674 Copenhagen V (DK) • JENSEN, Kim Birkebæk (73) Proprietor: Nuevolution A/S DK-2610 Rødovre (DK) 2100 Copenhagen 0 (DK) • PETERSEN, Lene DK-2100 Copenhagen Ø (DK) (72) Inventors: • NØRREGAARD-MADSEN, Mads • FRANCH, Thomas DK-3460 Birkerød (DK) DK-3070 Snekkersten (DK) • GODSKESEN,
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
    [Show full text]
  • Transcriptomic and Proteomic Profiling Provides Insight Into
    BASIC RESEARCH www.jasn.org Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy † † ‡ Peidi Liu,* Emelie Lassén,* Viji Nair, Celine C. Berthier, Miyuki Suguro, Carina Sihlbom,§ † | † Matthias Kretzler, Christer Betsholtz, ¶ Börje Haraldsson,* Wenjun Ju, Kerstin Ebefors,* and Jenny Nyström* *Department of Physiology, Institute of Neuroscience and Physiology, §Proteomics Core Facility at University of Gothenburg, University of Gothenburg, Gothenburg, Sweden; †Division of Nephrology, Department of Internal Medicine and Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; ‡Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan; |Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and ¶Integrated Cardio Metabolic Centre, Karolinska Institutet Novum, Huddinge, Sweden ABSTRACT IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type–specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell–positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell–positive standard genes.
    [Show full text]
  • Shared Sulfur Mobilization Routes for Trna Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes
    biomolecules Review Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes Silke Leimkühler *, Martin Bühning and Lena Beilschmidt Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany; [email protected] (M.B.); [email protected] (L.B.) * Correspondence: [email protected]; Tel.: +49-331-977-5603 Academic Editor: Valérie de Crécy-Lagard Received: 8 December 2016; Accepted: 9 January 2017; Published: 14 January 2017 Abstract: Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm5s2U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron–sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes. Keywords: tRNA; molybdenum cofactor; persulfide; thiocarboxylate; thionucleosides; sulfurtransferase; L-cysteine desulfurase 1.
    [Show full text]
  • PNAS Plus Significance Statements PNAS PLUS
    PNAS PLUS PNAS Plus Significance Statements PNAS PLUS Recombinant silicateins as model biocatalysts in properties, occurring on attosecond timescales on up − organosiloxane chemistry (1 as = 10 18 s). The recent development of attosecond S. Yasin Tabatabaei Dakhili, Stephanie A. Caslin, Abayomi S. angle-resolved photoemission spectroscopy (atto- Faponle, Peter Quayle, Sam P. de Visser, and Lu Shin Wong ARPES) using high harmonic generation has opened – Organosiloxanes are components in a huge variety of up the possibility of probing electron electron interact- – consumer products and play a major role in the syn- ions in real time. In this paper, we distinguish electron thesis of fine chemicals. However, their synthetic electron screening and charge scattering in the time manipulation primarily relies on the use of chloro- domain in individual energy bands within a solid. silanes, which are energy-intensive to produce and These results open up new possibilities for probing – environmentally undesirable. Synthetic routes that fundamental electron electron interactions in a host of operate under ambient conditions and circumvent the materials including magnetic, superconducting, and – need for chlorinated feedstocks would therefore offer advanced quantum materials. (See pp. E5300 E5307.) a more sustainable route for producing this class of compounds. Here, a systematic survey is reported for Application of metabolic controls for the the silicatein enzyme, which is able to catalyze the maximization of lipid production in hydrolysis, condensation, and exchange of the sili- semicontinuous fermentation con–oxygen bond in a variety of organosiloxanes Jingyang Xu, Nian Liu, Kangjian Qiao, Sebastian Vogg, under environmentally benign conditions. These re- and Gregory Stephanopoulos sults suggest that silicatein is a promising candidate This work establishes a semicontinuous process for for development of selective and efficient biocatalysts efficient and complete upgrading of low-strength for organosiloxane chemistry.
    [Show full text]
  • Structure and Functional Dynamics of the Mitochondrial Fe/S Cluster Synthesis Complex
    ARTICLE DOI: 10.1038/s41467-017-01497-1 OPEN Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex Michal T. Boniecki1, Sven A. Freibert 2, Ulrich Mühlenhoff2, Roland Lill 2,3 & Miroslaw Cygler 1,4 Iron–sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine 1234567890 desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status. 1 Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5. 2 Institut für Zytobiologie und Zytopathologie, Philipps-Universität, Robert-Koch-Strasse 6, 35032 Marburg, Germany. 3 LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans- Meerwein-Strasse, 35043 Marburg, Germany. 4 Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, Canada H3G 0B1.
    [Show full text]
  • Functional Characterization of the Eukaryotic Cysteine Desulfurase
    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 35, Issue of August 27, pp. 36906–36915, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Functional Characterization of the Eukaryotic Cysteine Desulfurase Nfs1p from Saccharomyces cerevisiae*□S Received for publication, June 11, 2004 Published, JBC Papers in Press, June 25, 2004, DOI 10.1074/jbc.M406516200 Ulrich Mu¨ hlenhoff‡, Janneke Balk‡, Nadine Richhardt‡, Jens T. Kaiser§, Katalin Sipos ¶, Gyula Kispal†¶, and Roland Lill‡ʈ From the ‡Institut fu¨ r Zytobiologie und Zytopathologie, Philipps-Universita¨t Marburg, Robert-Koch Strasse 6, 35033 Marburg, Germany, §California Institute of Technology, M/C 114–96, Pasadena, California 91125, and ¶Institute of Biochemistry, Medical Faculty, University of Pecs, Szigeti ut 12, 7624 Pecs, Hungary Previous studies have indicated that the essential pro- fur from cysteine via a cysteine persulfide intermediate that is tein Nfs1 performs a crucial role in cellular iron-sulfur bound to an essential cysteine residue close to the C terminus (Fe/S) protein maturation. The protein is located pre- (3). Sulfur abstraction involves the transient binding of the dominantly in mitochondria, yet low amounts are pres- cysteine substrate to the PLP cofactor via a Schiff base. NifS- ent in cytosol and nucleus. Here we examined several like proteins are structured into two domains, the larger bear- aspects concerning the molecular function of yeast ing the PLP co-factor and the smaller hosting the active site Nfs1p as a model protein. First, we demonstrated that cysteine residue in the middle of a highly flexible loop (4, 5).
    [Show full text]
  • The Cysteine-Desulfurase Iscs Promotes the Production of the Rhodanese Rhda in the Persulfurated Form
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 579 (2005) 6786–6790 FEBS 30176 The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form Fabio Forlania, Angelo Ceredaa, Andrea Freuerb, Manfred Nimtzc, Silke Leimku¨hlerd, Silvia Pagania,* a Dipartimento di Scienze Molecolari Agroalimentari, Facolta` di Agraria, Universita` di Milano, Via Celoria 2, Milano 20133, Italy b Institut fu¨r Pflanzenbiologie, TU Braunschweig, 38106 Braunschweig, Germany c Institut fu¨r Strukturbiologie, Gesellschaft fu¨r Biotechnologische Forschung (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany d Institut fu¨r Biochemie und Biologie, Universita¨t Potsdam, 14476 Potsdam, Germany Received 20 September 2005; revised 3 November 2005; accepted 5 November 2005 Available online 21 November 2005 Edited by Miguel De la Rosa Azotobacter vinelandii RhdA [17] appears to be a unique Abstract After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfu- member of the rhodanese homology superfamily [1,18,19]. rated form (RhdA-SSH). We identified L-cysteine as the most The RhdA active-site motif (HCQTHHR) is not currently effective sulfur source in producing RhdA-SSH. An E. coli solu- found in rhodanese-like proteins, and generates an unusually ble extract was required for in vitro persulfuration of RhdA, and strong positive electrostatic field [1] that favors stabilization the addition of pyridoxal-50-phosphate increased RhdA-SSH of a persulfide bond on the catalytic cysteine residue production, indicating a likely involvement of a cysteine desulfur- (Cys230), the only cysteine residue in this protein.
    [Show full text]
  • Shared Sulfur Mobilization Routes for Trna Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes
    Mathematisch-Naturwissenschaftliche Fakultät Silke Leimkühler | Martin Bühning | Lena Beilschmidt Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes Suggested citation referring to the original publication: Biomolecules 7 (2017) 1, Art. 5 DOI: 10.3390/biom7010005 DOI https://doi.org/10.3390/biom7010005 ISSN (online) 2218-273X Postprint archived at the Institutional Repository of the Potsdam University in: Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe ; 1015 ISSN 1866-8372 https://nbn-resolving.org/urn:nbn:de:kobv:517-opus4-475011 DOI https://doi.org/10.25932/publishup-47501 biomolecules Review Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes Silke Leimkühler *, Martin Bühning and Lena Beilschmidt Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany; [email protected] (M.B.); [email protected] (L.B.) * Correspondence: [email protected]; Tel.: +49-331-977-5603 Academic Editor: Valérie de Crécy-Lagard Received: 8 December 2016; Accepted: 9 January 2017; Published: 14 January 2017 Abstract: Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications
    [Show full text]
  • Mechanism of Activation of the Human Cysteine Desulfurase Complex by Frataxin
    Mechanism of activation of the human cysteine desulfurase complex by frataxin Shachin Patraa and David P. Barondeaua,1 aDepartment of Chemistry, Texas A&M University, College Station, TX 77842 Edited by Dennis R. Dean, Virginia Polytechnic Institute, Blacksburg, VA, and accepted by Editorial Board Member Angela M. Gronenborn August 13, 2019 (received for review June 6, 2019) The function of frataxin (FXN) has garnered great scientific interest and function of the NFS1 catalytic subunit (13–18). Both NFS1 since its depletion was linked to the incurable neurodegenerative and IscS generate a persulfide intermediate on a conserved cysteine disease Friedreich’s ataxia (FRDA). FXN has been shown to be nec- residue of a mobile S-transfer loop; the sulfur is then transferred essary for iron-sulfur (Fe-S) cluster biosynthesis and proper mito- to a cysteine residue on the Fe-S scaffold protein (19). In humans, + chondrial function. The structural and functional core of the Fe-S the scaffold protein ISCU2 combines the sulfane sulfur with Fe2 + cluster assembly complex is a low-activity pyridoxal 5′-phosphate and electrons, possibly from a ferredoxin (20, 21), to generate [2Fe-2S]2 (PLP)–dependent cysteine desulfurase enzyme that consists of cat- cluster intermediates (5, 22, 23). These clusters are then trans- alytic (NFS1), LYRM protein (ISD11), and acyl carrier protein (ACP) ferred intact to a carrier protein to complete the catalytic cycle for subunits. Although previous studies show that FXN stimulates the the assembly complex. activity of this assembly complex, the mechanism of FXN activa- Identifying the role of FXN in Fe-S cluster biosynthesis has tion is poorly understood.
    [Show full text]
  • Functional Investigation of Bacillus Subtilis Yrkf's Involvement in Sulfur
    Peptidomics 2015; 2: 45–51 Original Study Open Access Hannah L. Martin, Katherine A. Black, Patricia C. Dos Santos* Functional investigation of Bacillus subtilis YrkF’s involvement in sulfur transfer reactions DOI 10.1515/ped-2015-0008 enzymes involved in the synthesis of the pterin organic Received August 6, 2015; accepted December 5, 2015 moiety, the insertion of sulfur and Mo to form Moco, Abstract: Sulfur incorporation into the molybdenum and the activation of their respective enzymatic partners cofactor (Moco) in the Gram-negative bacterium [4]. While components in its biosynthetic pathway vary Escherichia coli involves six enzymes. The initial between species, the structure and functionality of the reaction includes the cysteine desulfurase IscS, the active cofactor is widely conserved [5]. sulfurtransferase TusA, and the rhodanese domain- The synthesis of Moco in the Gram-negative containing protein YnjE. The Gram-positive bacterium bacterium Escherichia coli is well understood [4]. The Bacillus subtilis contains no direct homologs for IscS, organic scaffolding is synthesized from guanosine but rather four distinct cysteine desulfurases (YrvO, NifS, triphosphate (GTP) via the intermediates cyclic NifZ, SufS) and YrkF, a two-domain rhodanese protein pyranopterin monophosphate (cPMP) and molybdopterin with an N-terminal domain similar to TusA. Bioinformatic (MPT) [6], which is subsequently adenylated prior to analysis was used to identify potential enzymes involved coordinating Mo [7,8]. The conversion of cPMP to MPT in the B. subtilis Moco thiolation pathway and in vitro requires the insertion of two sulfur atoms [9], constituting reactions demonstrated that YrkF can accept sulfur from the thiolation branch of Moco biosynthesis (Figure 1).
    [Show full text]