DOCSLIB.ORG

Dependent Enzyme Moaa and Its Implications for Molybdenum Cofactor Deficiency in Humans

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Dependent Enzyme Moaa and Its Implications for Molybdenum Cofactor Deficiency in Humans Crystal structure of the S-adenosylmethionine- dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans Petra Ha¨ nzelmann and Hermann Schindelin* Department of Biochemistry and Center for Structural Biology, State University of New York, Stony Brook, NY 11794-5115 Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved July 21, 2004 (received for review June 28, 2004) The MoaA and MoaC proteins catalyze the first step during molyb- neously occupied by N and O atoms from the methionine moiety denum cofactor biosynthesis, the conversion of a guanosine de- of the cofactor. rivative to precursor Z. MoaA belongs to the S-adenosylmethionine MoaA shares 14% and 11% identity in the N-terminal region (SAM)-dependent radical enzyme superfamily, members of which with BioB and HemN, respectively, but is completely unrelated catalyze the formation of protein and͞or substrate radicals by with these proteins in the C-terminal region, which is in MoaA reductive cleavage of SAM by a [4Fe–4S] cluster. A defined in vitro characterized by another Cys-rich signature motif. Recently, it system is described, which generates precursor Z and led to the could be shown that human MOCS1A in fact assembles two -identification of 5؅-GTP as the substrate. The structures of MoaA in oxygen-sensitive [4Fe–4S] clusters, one typical for SAM the apo-state (2.8 Å) and in complex with SAM (2.2 Å) provide dependent radical enzymes and an additional one unique to valuable insights into its mechanism and help to define the defects MoaA proteins (4). The structure of MoaC has been determined caused by mutations in the human ortholog of MoaA that lead to earlier, and the protein was found to be present as a hexamer molybdenum cofactor deficiency, a usually fatal disease accompa- composed of three dimers with a putative active site located at nied by severe neurological symptoms. The central core of each the dimer interface (12). Because some of the SAM-dependent subunit of the MoaA dimer is an incomplete triosephosphate radical enzymes require another protein onto which the radical isomerase barrel formed by the N-terminal part of the protein, is transferred, it has been speculated that MoaC might act in a which contains the [4Fe–4S] cluster typical for SAM-dependent similar function. Here, we describe the structure of MoaA, the radical enzymes. SAM is the fourth ligand to the cluster and binds final player in the Moco biosynthetic pathway (13). In addition, to its unique Fe as an N͞O chelate. The lateral opening of the an in vitro system for precursor Z synthesis is reported, which led incomplete triosephosphate isomerase barrel is covered by the to the identification of 5Ј-GTP as the substrate. C-terminal part of the protein containing an additional [4Fe–4S] cluster, which is unique to MoaA proteins. Both FeS clusters are Methods separated by Ϸ17 Å, with a large active site pocket between. The Cloning, Expression, and Purification. See Supporting Text, which is noncysteinyl-ligated unique Fe site of the C-terminal [4Fe–4S] published as supporting information on the PNAS web site. cluster is proposed to be involved in the binding and activation of 5؅-GTP. Crystallization of MoaA. Crystals were grown under anaerobic conditions inside a glove box (Coy Laboratory Products, Ann Ͻ n humans, genetic deficiencies of enzymes involved in molyb- Arbor, MI) containing 2 ppm O2 with the hanging drop vapor diffusion technique by incubating MoaA (34 mg͞ml, 100 mM Idenum cofactor (Moco) biosynthesis lead to the pleiotropic ⅐ ͞ loss of the molybdoenzymes sulfite oxidase, aldehyde oxidase, Tris HCl, pH 9.0 300 mM NaCl) with 10 mM DTT for 30 min and xanthine dehydrogenase and trigger an autosomal recessive on ice and subsequent mixing in a 1:1 ratio with precipitant solution [90 mM Na Hepes, pH 7.5͞3.15 M Na formate͞3% and usually fatal disease, which is characterized by severe ͞ neurological symptoms (1, 2). The first step in Moco biosynthe- (vol vol) DMSO]. Crystals appeared within 2 days and were cryoprotected by soaking in mother liquor containing 30% sis, the conversion of a guanosine derivative to precursor Z, an ͞ (vol vol) glycerol. They belong to space group P212121 with cell oxygen-sensitive 6-alkyl pterin with a cyclic phosphate, is cata- ϭ ϭ ϭ lyzed by MoaA and MoaC (MOCS1A and MOCS1B in humans) dimensions of a 48.1, b 102.4, and c 191.2 Å and contain (3, 4). As in the pathways of folate, riboflavin, and biopterin two molecules per asymmetric unit (57% solvent content). synthesis, a guanosine derivative serves as the initial biosynthetic precursor (5, 6), but in contrast to all other pathways, its C8 atom Data Collection and Structure Determination. Fe–multiwavelength is retained and incorporated in a rearrangement reaction as the anomalous diffraction data were collected on a single crystal on first carbon of the precursor Z side chain. a Rigaku (Tokyo) RU-H3R rotating-anode x-ray generator MoaA belongs to the family of S-adenosylmethionine (SAM)- equipped with double focusing mirror optics and an R axis II dependent radical enzymes, members of which catalyze the imaging plate detector at a wavelength of 1.5418 Å and on formation of protein and͞or substrate radicals by reductive beamline X26C at the National Synchrotron Light Source at cleavage of SAM by a [4Fe–4S] cluster (7–9). Members of this Brookhaven National Laboratory (Upton, NY) on a Quantum family are involved in various metabolic processes, but until now 4R ADSC charge-coupled device detector at a wavelength of only two members have been structurally characterized: Biotin synthase (BioB) (10), which converts dethiobiotin to biotin and This paper was submitted directly (Track II) to the PNAS office. coproporphyrinogen III oxidase (HemN) (11), which catalyzes Abbreviations: SAM, S-adenosylmethionine; 5Ј-dA, 5Ј-deoxyadenosine; TIM, triosephos- the conversion of coproporphyrinogen III to protoporphyrino- phate isomerase; BioB, biotin synthase; HemN, coproporphyrinogen III oxidase; Moco, gen IX during heme biosynthesis. These enzymes display related molybdenum cofactor; MOCS, molybdenum cofactor synthesis. folds in their N-terminal regions, which also harbor the oxygen- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, labile [4Fe–4S] cluster. This cluster is ligated by only three Cys www.pdb.org (PDB ID codes 1TV7 and 1TV8). residues in the apoenzyme, whereas in the complex with SAM, *To whom correspondence should be addressed. E-mail: [email protected]. the vacant coordination site on one of the Fe atoms is simulta- © 2004 by The National Academy of Sciences of the USA 12870–12875 ͉ PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404624101 Downloaded by guest on October 1, 2021 1.7406 Å (Fe peak). All data sets were indexed, integrated, and scaled with HKL software (14). For subsequent calculations, the CCP4 suite was used (15) with exceptions as indicated. Four Fe sites were located with SOLVE (16), which was also used for phase refinement. Phases were subsequently improved with RESOLVE (17), and a model was built with the program O (18). The second monomer was generated with the NCS operation. The structure was refined with REFMAC5 incorporating translation, libration, and screw-rotation displacement (TLS) refinement in all cycles (19, 20). Each monomer was refined separately, and solvent molecules were automatically added with ARP (21). The MoaA–SAM complex structure was obtained by soaking crystals cocrystallized with 5 mM SAM in precipitant solution containing 10 mM SAM and 30% (vol͞vol) glycerol for 30 min. Data were collected on beamline X26C at the National Syn- chrotron Light Source, Brookhaven National Laboratory at a wavelength of 1.1 Å. The structure was solved by difference Fourier techniques and refined as described for apo-MoaA. Information concerning data collection and refinement sta- tistics is available in Tables 1 and 2, which are published as supporting information on the PNAS web site. Figures were created with MOLSCRIPT (22), BOBSCRIPT (23), PYMOL (DeLano Scientific, San Carlos, CA), SPOCK (24), and RASTER3D (25). Assays of Precursor Z Synthesizing Activity and Reductive Cleavage of SAM. Nitrate reductase overlay assays and in vivo assays in moaA (KB2037) and moaC (KB2066) mutant cells were conducted as Fig. 1. In vitro synthesis of precursor Z. (A Left) Synthesis of precursor Z determined by measurement of its stable oxidized derivative, compound Z, by described (3). In vitro assays were performed under anaerobic BIOCHEMISTRY reversed-phase HPLC. MoaA (50 ␮M) and MoaC (50 ␮M) were incubated under conditions at room temperature in a total volume of 180 ␮lof100 ⅐ ͞ anaerobic conditions in the presence of 2 mM DTT with a 10-fold molar excess mM Tris HCl, pH 9.0 300 mM NaCl in the presence of 2 mM Ј Ј of SAM, 5 -GTP, MgCl2, and Na2S2O4 for 45 min (standard assay). (Top to DTT and a 10-fold molar excess of SAM, 5 -GTP, MgCl2, bottom) Reconstituted MoaA; anaerobically purified MoaA; without MoaC. ␮ Na2S2O4, and 50 M MoaC. The reaction was started by adding (Right) Time dependence of precursor Z formation (F) and reductive cleavage 50 ␮M MoaA and terminated, at specified times, by the addition of SAM in the presence of 5Ј-GTP generating 5Ј-dA (E). (B) Requirements for of 220 ␮l 100 mM Tris⅐HCl, pH 7.2͞50 ␮l of acidic iodine to precursor Z synthesis. The maximal amount of compound Z formed in a convert precursor Z to its stable fluorescent product compound standard assay (control, see A) was set to 100%. 5Ј-GTP was absent from the Z. After incubation at room temperature for 14 h, compound Z experiment with 5Ј-GDP and 5Ј-GMP.
Load more

Recommended publications
  • Crystallographic Snapshots of Sulfur Insertion by Lipoyl Synthase
    Crystallographic snapshots of sulfur insertion by lipoyl synthase Martin I. McLaughlina,b,1, Nicholas D. Lanzc, Peter J. Goldmana, Kyung-Hoon Leeb, Squire J. Bookerb,c,d, and Catherine L. Drennana,e,f,2 aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802; cDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802; dHoward Hughes Medical Institute, The Pennsylvania State University, University Park, PA 16802; eDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and fHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Vern L. Schramm, Albert Einstein College of Medicine, Bronx, NY, and approved July 5, 2016 (received for review March 8, 2016) Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms substrate and at an intermediate stage in the reaction, just after at the unactivated C6 and C8 positions of a protein-bound octanoyl insertion of the C6 sulfur atom but before sulfur insertion at C8. chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethio- Results nine (AdoMet) radical chemistry; the remainder of the reaction The crystal structure of LipA from M. tuberculosis was de- mechanism, especially the source of the sulfur, has been less clear. termined to 1.64-Å resolution by iron multiwavelength anoma- One controversial proposal involves the removal of sulfur from a lous dispersion phasing (Table S1). The overall fold of LipA consists second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in de- of a (β/α)6 partial barrel common to most AdoMet radical enzymes struction of the cluster during each round of catalysis.
    [Show full text]
  • Letters to Nature
    letters to nature Received 7 July; accepted 21 September 1998. 26. Tronrud, D. E. Conjugate-direction minimization: an improved method for the re®nement of macromolecules. Acta Crystallogr. A 48, 912±916 (1992). 1. Dalbey, R. E., Lively, M. O., Bron, S. & van Dijl, J. M. The chemistry and enzymology of the type 1 27. Wolfe, P. B., Wickner, W. & Goodman, J. M. Sequence of the leader peptidase gene of Escherichia coli signal peptidases. Protein Sci. 6, 1129±1138 (1997). and the orientation of leader peptidase in the bacterial envelope. J. Biol. Chem. 258, 12073±12080 2. Kuo, D. W. et al. Escherichia coli leader peptidase: production of an active form lacking a requirement (1983). for detergent and development of peptide substrates. Arch. Biochem. Biophys. 303, 274±280 (1993). 28. Kraulis, P.G. Molscript: a program to produce both detailed and schematic plots of protein structures. 3. Tschantz, W. R. et al. Characterization of a soluble, catalytically active form of Escherichia coli leader J. Appl. Crystallogr. 24, 946±950 (1991). peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 34, 3935±3941 29. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and (1995). the thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281±296 (1991). 4. Allsop, A. E. et al.inAnti-Infectives, Recent Advances in Chemistry and Structure-Activity Relationships 30. Meritt, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505± (eds Bently, P. H. & O'Hanlon, P. J.) 61±72 (R. Soc. Chem., Cambridge, 1997).
    [Show full text]
  • Mitochondrial Serine Acetyltransferase Functions As a Pacemaker of Cysteine Synthesis in Plant Cells1[C][W][OA]
    Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells1[C][W][OA] Florian H. Haas, Corinna Heeg, Rafael Queiroz, Andrea Bauer, Markus Wirtz, and Ru¨ diger Hell* Heidelberg Institute for Plant Sciences, Heidelberg University, 69120 Heidelberg, Germany (F.H.H., C.H., M.W.,R.H.);andGermanCancerResearchCenter,69120Heidelberg,Germany(R.Q.,A.B.) Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide.
    [Show full text]
  • Product Sheet Info
    Master Clone List for NR-19274 Mycobacterium tuberculosis Gateway® Clone Set, Recombinant in Escherichia coli, Plates 1-42 Catalog No. NR-19274 Table 1: Mycobacterium tuberculosis, Gateway® Clones, Plate 1 (ZMTDA), NR-19637 Clone Well ORF Locus ID Description (Gene name) Accession Average Depth Position Length Number of Coverage 71201 A01 124 Rv1572c hypothetical protein Rv1572c NP_216088.2 2 71005 A02 151 Rv3461c 50S ribosomal protein L36 (rpmJ) NP_217978.1 2 71053 A03 181 Rv3924c 50S ribosomal protein L34 (rpmH) 2 71013 A04 184 Rv2452c hypothetical protein Rv2452c NP_216968.1 2 71167 A05 193 Rv0657c hypothetical protein Rv0657c NP_215171.1 2.69948187 71177 A06 211 Rv0666 hypothetical protein Rv0666 NP_215180.1 2 71225 A07 214 Rv1693 hypothetical protein Rv1693 NP_216209.1 2 71073 A08 217 Rv2099c PE family protein (PE21) 2 70874 A09 220 Rv0810c hypothetical protein Rv0810c NP_215325.1 2 70913 A10 223 Rv2371 PE-PGRS family protein (PE_PGRS40) YP_177875.1 2 71141 A11 229 Rv2806 hypothetical protein Rv2806 NP_217322.1 2 71121 A12 235 Rv1113 hypothetical protein Rv1113 NP_215629.1 1.99574468 71181 B01 241 Rv3648c cold shock protein A (cspA) NP_218165.1 2 70937 B02 244 Rv0763c ferredoxin NP_215277.1 2 70966 B03 247 Rv1054 integrase NP_215570.2 1.27530364 71145 B04 253 Rv2377c putative protein MbtH (mbtH) NP_216893.1 2 70861 B05 253 Rv2830c hypothetical protein Rv2830c NP_217346.1 2 70853 B06 253 Rv3221c anti-sigma factor YP_177945.1 2 71210 B07 256 Rv1893 hypothetical protein Rv1893 NP_216409.1 2 71062 B08 259 Rv0378 glycine rich protein
    [Show full text]
  • The Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme F
    2-74 LIFE SCIENCES SCIENCE HIGHLIGHTS 2-75 The Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme F. Berkovitch1, Y. Nicolet1, J.T. Wan2, J.T. Jarrett2, and C.L. Drennan1 1Department of Chemistry, Massachusetts Institute of Technology; 2Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania BEAMLINE X25 The crystal structure of biotin synthase addresses how “AdoMet radical” enzymes, also called “Radical SAM” enzymes, use an Fe S cluster and S-adenosyl-L-methionine to 4 4 Funding generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of National Institutes of Health; Searle Scholars Program; sulfur into dethiobiotin (DTB) to form biotin (vitamin B8). The structure places the substrates, i.e. DTB and AdoMet, between the Fe S cluster (essential for radical gen- Cecil and Ida Green Career 4 4 Development Fund; Lester eration) and the Fe2S2 cluster (postulated to be the source of sulfur), with both clusters Wolfe Predoctoral Fellowship; in unprecedented coordination environments. Cellular, Biochemical, and Molecular Sciences training Biotin is an essential vitamin that plays a ubiquitous role in human growth and grant; U.S. Department of Energy; National Institute of metabolism. Biotin deficiency results in skin lesions, abnormal fat distribution, General Medical Sciences neurological symptoms, and immunodeficiency. A low biotin level has also been correlated to an increased incidence of type II diabetes mellitus. Biotin is a valuable Publication commercial commodity, used as an additive in food, health, and cosmetic prod- F. Berkovitch, Y. Nicolet, J.T. Wan, J.T. Jarrett, and ucts, and as a research tool in the biochemical sciences.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Radical-Sam Enzymes with Two Iron-Sulfur Clusters: Cofactor
    RADICAL-SAM ENZYMES WITH TWO IRON-SULFUR CLUSTERS: COFACTOR COMPOSITION AND SPECTROSCOPIC STUDIES OF ESCHERICHIA COLI AND BACILLUS SUBTILIS BIOTIN SYNTHASE, HUMAN MOCS1A, AND THERMOTOGA MARITIMA MIAB by HEATHER LOUISE HERNÁNDEZ (Under the Direction of Michael Kenneth Johnson) ABSTRACT A new class of Fe-S proteins, termed radical-SAM enzymes, catalyzes radical reactions in a variety of biosynthetic processes. These [4Fe-4S]2+,+ cluster-containing enzymes initiate radical enzymatic reactions via reductive cleavage of S-adenosyl-L-methionine (SAM) to yield methionine and an extremely reactive 5'-deoxyadenosyl radical. A growing number of radical-SAM enzymes have recently been discovered to contain a second Fe-S cluster of unknown function, although possible roles include acting as sacrificial S-donor or anchoring and possibly activating the substrate. The combination of analytical and spectroscopic studies, including EPR, Mössbauer, UV-visible absorption/circular dichroism/variable temperature magnetic circular dichroism, and resonance Raman, have been used to investigate the cofactor composition and properties of the two cluster containing radical-SAM enzymes Escherichia coli and Bacillus subtilis biotin synthase (BioB), human MOCS1A, and Thermotoga maritima MiaB. These enzymes are involved in crucial steps in the biosynthesis of biotin and molybdopterin, and in the thiomethylation of tRNA. E. coli and B. subtilis BioB are shown to house a radical-SAM [4Fe-4S] cluster and a [2Fe-2S] cluster in separate binding sites. The function and relevance of the [2Fe-2S] cluster, which has been suggested to be the S-donor to biotin, is addressed. In E. coli BioB, the most active form of the enzyme contains a 1:1 ratio of [2Fe-2S]/[4Fe-4S] clusters and the [2Fe-2S] cluster degrades during turnover.
    [Show full text]
  • Cysteine Catabolism and Glucosinolate Turnover in Arabidopsis Thaliana
    Cysteine catabolism and glucosinolate turnover in Arabidopsis thaliana Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von Saskia Brandt geb. Höfler, M. Sc. 2018 Referent: Prof. Dr. rer. nat. Hans-Peter Braun Korreferent: Prof. Dr. rer. nat. Helge Küster Tag der Promotion: 14.02.2018 The following publications contributed to this thesis: 1. Höfler S., Lorenz C., Busch T., Brinkkötter M., Tohge T., Fernie A.R., Braun H.-P., and Hildebrandt T.M. (2016): Dealing with the sulfur part of cysteine: four enzymatic steps degrade L-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria. Physiologia Plantarum 157 (3): 255–399 2. Lorenz C., Brandt S., Borisjuk L., Rolletschek H., Heinzel N., Tohge T., Fernie A.R., Braun H.-P., Hildebrandt T.M. (in preparation): Amino acid metabolism and the role of ETHE1 in Arabidopsis thaliana seeds. 3. Brandt S., Fachinger S., Tohge T., Fernie A.R., Braun H.-P. and Hildebrandt T.M. (in preparation): Extended darkness induces internal turnover of glucosinolates in Arabidopsis leaves. I Abstract Sulfur is an important chemical element in plants. It is taken up in form of sulfate and assimilation takes place in the cytosol, mitochondria and chloroplasts. The result of this process is, among others, the amino acid cysteine and the secondary metabolite glucosinolate. Under abiotic stress conditions such as low light or extended darkness, the plant has to catabolize amino acids and other metabolites to gain nutrients and energy. Cysteine can be catabolized via three pathways to pyruvate and persulfide.
    [Show full text]
  • Cysteine: an Overlooked Energy and Carbon Source
    www.nature.com/scientificreports OPEN Cysteine: an overlooked energy and carbon source Luise Göbbels1, Anja Poehlein2, Albert Dumnitch1, Richard Egelkamp2, Cathrin Kröger1, Johanna Haerdter3, Thomas Hackl3, Artur Feld4, Horst Weller4, Rolf Daniel2, Wolfgang R. Streit1 & Marie Charlotte Schoelmerich1* Biohybrids composed of microorganisms and nanoparticles have emerged as potential systems for bioenergy and high-value compound production from CO2 and light energy, yet the cellular and metabolic processes within the biological component of this system are still elusive. Here we dissect the biohybrid composed of the anaerobic acetogenic bacterium Moorella thermoacetica and cadmium sulphide nanoparticles (CdS) in terms of physiology, metabolism, enzymatics and transcriptomic profling. Our analyses show that while the organism does not grow on l-cysteine, it is metabolized to acetate in the biohybrid system and this metabolism is independent of CdS or light. CdS cells have higher metabolic activity, despite an inhibitory efect of Cd2+ on key enzymes, because of an intracellular storage compound linked to arginine metabolism. We identify diferent routes how cysteine and its oxidized form can be innately metabolized by the model acetogen and what intracellular mechanisms are triggered by cysteine, cadmium or blue light. Anaerobic microorganisms play a pivotal role in the global cycling of elements and nutrients, are key in determin- ing the health of animals and humans and are important players for biotechnological and industrial processes to solve tomorrow’s challenges of pollution, resource scarcity and energy requirements 1,2. An innovative proof-of- concept study has recently been described in which a non-photosynthetic anaerobic bacterium becomes photo- synthetic by coupling it to CdS nanoparticles3.
    [Show full text]
  • Cluster Accompanies Formation of the Intermediate 9-Mercaptodethiobiotin in Escherichia Coli Biotin Synthase Andrew M
    Article pubs.acs.org/biochemistry Reduction of the [2Fe−2S] Cluster Accompanies Formation of the Intermediate 9-Mercaptodethiobiotin in Escherichia coli Biotin Synthase Andrew M. Taylor, Stefan Stoll,! R. David Britt,! and Joseph T. Jarrett*, Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States !Department of Chemistry, University of California, Davis, Davis, California 95616, United States ABSTRACT: Biotin synthase catalyzes the conversion of dethiobiotin (DTB) to biotin through the oxidative addition of sulfur between two saturated carbon atoms, generating a thiophane ring fused to the existing ureido ring. Biotin synthase is a member of the radical SAM superfamily, composed of enzymes that reductively cleave S-adenosyl-L-methionine (SAM or AdoMet) to generate a 52-deoxyadenosyl radical that can abstract unactivated hydrogen atoms from a variety of organic substrates. In biotin synthase, abstraction of a hydrogen atom from the C9 methyl group of DTB would result in formation of a dethiobiotinyl methylene carbon radical, which is then quenched by a sulfur atom to form a new carbon−sulfur bond in the intermediate 9-mercaptodethiobiotin (MDTB). We have proposed that this sulfur atom is the ¼-sulfide of a [2Fe−2S]2+ cluster found near DTB in the enzyme active site. In the present work, we show that formation of MDTB is accompanied by stoichiometric generation of a paramagnetic FeS cluster. The electron paramagnetic resonance (EPR) spectrum is modeled as a 2:1 mixture of components attributable to different forms of a [2Fe−2S]+ cluster, possibly distinguished by slightly different coordination environments. Mutation of Arg260, one of the ligands to the [2Fe−2S] cluster, causes a distinctive change in the EPR spectrum.
    [Show full text]
  • Genetic and Biochemical Characterization of Yrkf, a Novel
    Genetic and biochemical characterization of YrkF, a novel two-domain sulfurtransferase in Bacillus subtilis Jeremy Hunt Thesis submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Biochemistry Timothy J. Larson, Chair Dennis R. Dean Brenda J. Winkel August 11, 2004 Blacksburg, Virginia Keywords: YrkF, rhodanese, Ccd1, persulfide sulfur, disulfide cross-linking Genetic and biochemical characterization of YrkF, a novel two-domain sulfurtransferase in Bacillus subtilis Jeremy Hunt Timothy J. Larson, Chair Department of Biochemistry ABSTRACT Sulfur-containing compounds such as thiamin, biotin, molybdopterin, lipoic acid, and [Fe-S] clusters are essential for life. Sulfurtransferases are present in eukaryotes, eubacteria, and archaea and are believed to play important roles in mobilizing sulfur necessary for biosynthesis of these compounds and for normal cellular functions. The rhodanese homology domain is a ubiquitous structural module containing a characteristic active site cysteine residue. Some proteins containing a rhodanese domain display thiosulfate:cyanide sulfurtransferase activity in vitro. However, the physiological functions of rhodaneses remain largely unknown. YrkF, the first rhodanese to be characterized from Bacillus subtilis, is a unique protein containing two domains, an N-terminal Ccd1 domain and a C-terminal rhodanese domain. Ccd1 (conserved cysteine domain 1) is a ubiquitous structural module characterized by a Cys-Pro-X-Pro sequence motif. Thus, YrkF contains two cysteine residues (Cys15 and Cys149), one in each domain. Biochemical, genetic, and bioinformatic approaches were used in order to characterize YrkF. First, YrkF was overexpressed and assayed for rhodanese activity to show that the protein is a functional rhodanese.
    [Show full text]
  • Genome Wide Analysis Identifies Sphingolipid Metabolism As a New Target of Valproic Acid" (2016)
    Wayne State University Wayne State University Dissertations 1-1-2016 Genome Wide Analysis Identifies phinS golipid Metabolism As A New Target Of Valproic Acid Shyamalagauri Jadhav Jadhav Wayne State University, Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Biology Commons, and the Molecular Biology Commons Recommended Citation Jadhav, Shyamalagauri Jadhav, "Genome Wide Analysis Identifies Sphingolipid Metabolism As A New Target Of Valproic Acid" (2016). Wayne State University Dissertations. 1545. https://digitalcommons.wayne.edu/oa_dissertations/1545 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. GENOME WIDE ANALYSIS IDENTIFIES SPHINGOLIPID METABOLISM AS A NEW TARGET OF VALPROIC ACID by SHYAMALAGAURI JADHAV DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2016 MAJOR: BIOLOGICAL SCIENCES Approved By: _____________________________ Advisor Date _____________________________ _____________________________ _____________________________ ©COPYRIGHT BY SHYAMALAGAURI JADHAV 2016 All Rights Reserved DEDICATION I would like to dedicate my dissertation to my late mother Chandrakala Jadhav who was a constant support and inspiration through the years. ii ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Dr. Miriam L. Greenberg for her intellectual, moral, financial, and emotional support throughout my graduate journey. The realization of this thesis would have been impossible without her constant encouragement and strong support. I would like to thank her for the confidence she had in my ability to think and design the project through; and for her guidance when needed.
    [Show full text]