DOCSLIB.ORG

Clostridium Tetanomorphum (Bilatriene/Uroporphyrin-Related/Enzymatic Formation/Purification/Characterization) PHILLIP J

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Clostridium Tetanomorphum (Bilatriene/Uroporphyrin-Related/Enzymatic Formation/Purification/Characterization) PHILLIP J Proc. NatL Acad. Sci. USA Vol. 80, pp. 3943-3947, July 1983 Biochemistry Bactobilin: Blue bile pigment isolated from Clostridium tetanomorphum (bilatriene/uroporphyrin-related/enzymatic formation/purification/characterization) PHILLIP J. BRUMM*t, JOSEF FRIED*t, AND HERBERT C. FRIEDMANN*§ Departments of *Biochemistry and tChemistry, The University of Chicago, Chicago, Illinois 60637 Contributed byJosef Fried, March 31, 1983 ABSTRACT A blue bile pigment, possessing four acetic and used were reagent grade or better. Silica gel for flash chro- four propionic acid side chains has been isolated from extracts of matography and a flash chromatography column, 3.4-cm di- the anaerobic microorganism Clostridium tetanomorphum and in ameter, were obtained from J. T. Baker. Whatman high-per- smaller amounts from Propionibacterium shermanii. The com- formance silica thin-layer chromatography plates with a pre- pound could be prepared in larger amounts by incubation of C. adsorbent spotting area (type LHP-K, 10 x 20 cm) and E. Merck tetanomorphum enzyme extracts with added 8-aminolevulinic acid. precoated cellulose thin-layer chromatography plates were ob- The ultraviolet-visible, infrared, and proton magnetic resonance tained from Anspec (Ann Arbor, MI). For fluorescence detec- spectra of the pigment indicate a chromophore of the biliverdin tion, a lamp emitting at 365 nm was used. C. tetanomorphum type. Field-desorption mass spectrometry of the purified methyl cells (ATCC 15920) were grown on a medium containing ester showed a strong molecular ion at m/e = 962. This corre- yeast sponds to the molecular weight expected for the octamethyl ester extract and monosodium glutamate (based on medium 163, of a bilatriene type of bile pigment structurally derived from uro- American Type Culture Collection) as described (18) and were porphyrin 1m or I. Of the five possible structures, two could be collected by centrifugation. Uroporphyrin and C-methylated eliminated by proton magnetic resonance spectroscopy. The name mono-, di-, and trimethylisobacteriochlorins (Factors I, II, and bactobilin is proposed for this previously unreported bile pigment. III) were isolated as their octamethyl esters by standard meth- ods (19, 20). Sirohydrochlorin (Factor II) octamethyl ester was The open-chain tetrapyrrole compounds known as bile pig- given by A. I. Scott (College Station, TX); dimethyl- and ments are widely distributed. They are found free or bound to trimethylisobacteriochlorin dilactone octamethyl esters were protein in mammals, birds, amphibians, reptiles, fish, mol- given by V. Koppenhagen (Braunschweig-Stockheim, Federal luscs, and insects and in algae and higher plants (for reviews, Republic of Germany). see refs. 1-9). As side chains or 1&carbon ring substituents, all Enzyme Preparation. An acetone powder was prepared from bile pigments described thus far have four methyl groups, two the bacteria as follows. Immediately after centrifugation, 170- propionic acid groups, and two vinyl groups, one or both of which 180 g of packed cells (obtained from 56 liters of growth me- can be isomerized to ethylidene (4, 10, 11) or reduced to ethyl dium) were uniformly suspended in 170-180 ml of ice-cold 2% (6, 10, 11). These substituents are arranged in the sequence 2-mercaptoethanol with the help of a nonaerating stirrer (Kraft found in protoporphyrin IX; in fact, all these bile pigments are Apparatus, distributed by Glas-Col Apparatus, Terre Haute, formed from protoheme (8, 12-14), whose oxidative breakdown IN). The cold suspension was added slowly with stirring to 1.7- to biliverdin is catalyzed by the enzyme heme oxygenase (15- 1.8 liters of acetone at -100C; the stirring was continued for 17). Bile pigments thus far have not been detected in prokary- 5 min at about 0C. The material, collected by suction filtration, otes. The present paper reports the isolation and in vitro for- was resuspended uniformly in 1.7-1.8 liters of cold acetone with mation of a bile pigment from the bacterium Clostridium nonaerating stirring, collected again by filtration, and trans- tetanomorphum. This anaerobic organism makes uroporphyrin- ferred to a round-bottom flask. The remaining acetone was re- ogen, the precursor of vitamin B-12, but not heme, protopor- moved in vacuo at room temperature with the aid of a rotary phyrinogen, or coproporphyrinogen. The isolated bile pigment evaporator. The powder could be stored over a desiccant at is of interest not only because of its detection in a prokaryote, -20'C for at least 3 months without significant activity loss. but also because its 13-carbon ring substituents, four acetic acid The average yield was 50 g (i.e., about 29% of the packed cell and four propionic acid groups, correspond to those of a uro- weight). For enzyme extraction, 30 g of powder was stirred for porphyrin and not of protoporphyrin. 10 min at room temperature without aeration in 300 ml of 100 mM Tris'HCI buffer, pH 8.0/0.2% 2-mercaptoethanol. The suspension was centrifuged for 20 min at 48,000 X g. MATERIALS AND METHODS Preparation of Bacterial Bile Pigment. To the clear super- Supplies. -Aminolevulinic acid was obtained from Sigma; natant solution, 60 mg of 8-aminolevulinic acid dissolved in 10 biliverdin IXa dimethyl ester and the fully esterified methyl ml of 100 mM Tris (pH 8.0) was added with gentle swirling. esters of uroporphyrin III, coproporphyrin III, protoporphyrin The mixture was incubated 18 hr in the dark at 370C. Precip- IX, and heptacarboxylporphyrin I, were from Porphyrin Prod- itated protein was removed by centrifugation for 20 min at 48,000 ucts (Logan, UT). The n-hexane used for flash chromatography X g. The uroporphyrins and related anionic substances in the was "95 + %" (Aldrich), whereas the n-hexane for thin-layer supernatant solution were esterified after retention on an anion chromatography was 99 mol % pure (Fisher). Other chemicals exchanger by a modification of the method of Bergmann et al. The publication costs of this article were defrayed in part by page charge t Present address: Moffett Tech. Ctr., Corn Products, P.O. Box 345, payment. This article must therefore be hereby marked "advertise- Summitt-Argo, IL 60501. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. § To whom reprint requests should be addressed. 3943 Downloaded by guest on September 27, 2021 3944 Biochemistry: Brumm et al. Proc. Natl. Acad. Sci. USA 80 (1983) (19): about 5 g of DEAE-Sephadex A-25 that had been equil- Table 1. Ultraviolet-visible and infrared absorption maxima ibrated with 100 mM potassium phosphate buffer (pH 7.4) was in chloroform added in aqueous suspension with slight swirling. The ion ex- Ratio Ratio changer was collected by filtration and washed three times with UV-Vis 380/ IR 1,730/ 250 ml of water and three times with 250 ml of methanol. To Ama,) nm 650 Pmax, cm- 1,700 esterify the substances retained on this anhydrous DEAE- 200 Biliverdin IXa 1,732 (ester C=O) Sephadex, ml of methanol/sulfuric acid, 90:10 (vol/vol), dimethyl 376, 642-670 3.76 1,695 (amide C=O) 0.76 was added. The vessel was tightly capped. After 18 hr at 370C, ester the supernatant solution was collected by filtration, and the Bactobilin 1,735 (ester C=O) DEAE-Sephadex was washed with a small volume of chloro- octamethyl 369,644 3.56 1,705 (amide C=O) 1.27 form. The esters were extracted into chloroform by agitating ester the filtrate with about 200 ml each of water and of chloroform. The reddish chloroform layer was collected and washed twice Vis, visible. with 200 ml of 0.1 M ammonia. In this process yellow material was removed. Three washes with 200 ml of water followed. The RESULTS chloroform was removed in vacuo at room temperature, and the The present work resulted from studies of intermediates in vi- residue was dissolved in 10 ml of chloroform. The material was tamin B-12 biosynthesis. After incubation of enzyme extracts subjected to flash chromatography on 180 ml of silica gel that of C. tetanomorphum with 6-aminolevulinic acid at pH 8 in the had been equilibrated with a mixture of n-hexane/2-propanol/ presence of 2-mercaptoethanol and workup by conventional methanol, 5:2:1 (vol/vol) (refs. 19, 21, 22; and the unpublished methods (19), it was found that when the reaction products in method of A. I. Scott was incorrectly quoted in ref. 21 re- but the form of their methyl esters were submitted to thin-layer peated correctly in refs. 19 Pressure was and 22). applied with chromatography on silica plates in a widely used solvent (24), nitrogen or argon. was The column treated with 400 ml of this a certain amount of pigmented material remained near the or- solvent, followed by 200 ml of n-hexane/2-propanol, 1:1.5 (vol/ igin. Thin-layer chromatography of this material in chloroform vol). The eluates were combined, and the solvent was removed containing from 2% to 3% methanol showed small amounts of in vacuo. a striking hitherto unreported nonfluorescent blue material. The solid residue was dissolved in 20 ml of chloroform and The first indication that this substance is a bile pigment and not to area applied the preadsorbent spotting of 10 Whatman LHP- a cyclic tetrapyrrole was provided by the fact that it gave a pos- K were silica plates, which then developed in dim light in chlo- itive Gmelin reaction (multiple, gradually developing colors upon roform/methanol, 100:3 (vol/vol). The solvent troughs were careful addition of concentrated nitric acid) (described by Tie- lined with saturation pads. The main component was rapidly demann and Gmelin in 1826; for recent references see refs. 2, moving uroporphyrin octamethyl ester. The blue bands (Rf 25, and 26).
Load more

Recommended publications
  • Significance and Implications of Vitamin B-12 Reaction Shema- ETH ZURICH VARIANT: Mechanisms and Insights
    Taylor University Pillars at Taylor University Student Scholarship: Chemistry Chemistry and Biochemistry Fall 2019 Significance and Implications of Vitamin B-12 Reaction Shema- ETH ZURICH VARIANT: Mechanisms and Insights David Joshua Ferguson Follow this and additional works at: https://pillars.taylor.edu/chemistry-student Part of the Analytical Chemistry Commons, Inorganic Chemistry Commons, Organic Chemistry Commons, Other Chemistry Commons, and the Physical Chemistry Commons CHEMISTRY THESIS SIGNIFICANCE AND IMPLICATIONS OF VITAMIN B-12 REACTION SCHEMA- ETH ZURICH VARIANT: MECHANISMS AND INSIGHTS DAVID JOSHUA FERGUSON 2019 2 Table of Contents: Chapter 1 6 Chapter 2 17 Chapter 3 40 Chapter 4 59 Chapter 5 82 Chapter 6 118 Chapter 7 122 Appendix References 3 Chapter 1 A. INTRODUCTION. Vitamin B-12 otherwise known as cyanocobalamin is a compound with synthetic elegance. Considering how it is composed of an aromatic macrocyclic corrin there are key features of this molecule that are observed either in its synthesis of in the biochemical reactions it plays a role in whether they be isomerization reactions or transfer reactions. In this paper the focus for the discussion will be on the history, chemical significance and total synthesis of vitamin B12. Even more so the paper will be concentrated one of the two variants of the vitamin B-12 synthesis, namely the ETH Zurich variant spearheaded by Albert Eschenmoser.Examining the structure as a whole it is observed that a large portion of the vitamin B12 is a corrin structure with a cobalt ion in the center of the macrocyclic part, and that same cobalt ion has cyanide ligands.
    [Show full text]
  • Magnesium-Protoporphyrin Chelatase of Rhodobacter
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1941-1944, March 1995 Biochemistry Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: Reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli (protoporphyrin IX/tetrapyrrole/chlorophyll/bacteriochlorophyll/photosynthesis) LUCIEN C. D. GIBSON*, ROBERT D. WILLOWSt, C. GAMINI KANNANGARAt, DITER VON WETTSTEINt, AND C. NEIL HUNTER* *Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, United Kingdom; and tCarlsberg Laboratory, Department of Physiology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Contributed by Diter von Wettstein, November 14, 1994 ABSTRACT Magnesium-protoporphyrin chelatase lies at Escherichia coli and demonstrate that the extracts of the E. coli the branch point of the heme and (bacterio)chlorophyll bio- transformants can convert Mg-protoporphyrin IX to Mg- synthetic pathways. In this work, the photosynthetic bacte- protoporphyrin monomethyl ester (20, 21). Apart from posi- rium Rhodobacter sphaeroides has been used as a model system tively identifying bchM as the gene encoding the Mg- for the study of this reaction. The bchH and the bchI and -D protoporphyrin methyltransferase, this work opens up the genes from R. sphaeroides were expressed in Escherichia coli. possibility of extending this approach to other parts of the When cell-free extracts from strains expressing BchH, BchI, pathway. In this paper, we report the expression of the genes and BchD were combined, the mixture was able to catalyze the bchH, -I, and -D from R. sphaeroides in E. coli: extracts from insertion of Mg into protoporphyrin IX in an ATP-dependent these transformants, when combined in vitro, are highly active manner.
    [Show full text]
  • Metabolic Versatility of the Nitrite-Oxidizing Bacterium Nitrospira
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira 2 marina and its proteomic response to oxygen-limited conditions 3 Barbara Bayer1*, Mak A. Saito2, Matthew R. McIlvin2, Sebastian Lücker3, Dawn M. Moran2, 4 Thomas S. Lankiewicz1, Christopher L. Dupont4, and Alyson E. Santoro1* 5 6 1 Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, 7 CA, USA 8 2 Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution, 9 Woods Hole, MA, USA 10 3 Department of Microbiology, IWWR, Radboud University, Nijmegen, The Netherlands 11 4 J. Craig Venter Institute, La Jolla, CA, USA 12 13 *Correspondence: 14 Barbara Bayer, Department of Ecology, Evolution and Marine Biology, University of California, 15 Santa Barbara, CA, USA. E-mail: [email protected] 16 Alyson E. Santoro, Department of Ecology, Evolution and Marine Biology, University of 17 California, Santa Barbara, CA, USA. E-mail: [email protected] 18 19 Running title: Genome and proteome of Nitrospira marina 20 21 Competing Interests: The authors declare that they have no conflict of interest. 22 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.02.185504; this version posted July 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • AOP 131: Aryl Hydrocarbon Receptor Activation Leading to Uroporphyria
    Organisation for Economic Co-operation and Development DOCUMENT CODE For Official Use English - Or. English 1 January 1990 AOP 131: Aryl hydrocarbon receptor activation leading to uroporphyria Short Title: AHR activation-uroporphyria This document was approved by the Extended Advisory Group on Molecular Screening and Toxicogenomics in June 2018. The Working Group of the National Coordinators of the Test Guidelines Programme and the Working Party on Hazard Assessment are invited to review and endorse the AOP by 29 March 2019. Magdalini Sachana, Administrator, Hazard Assessment, [email protected], +(33- 1) 85 55 64 23 Nathalie Delrue, Administrator, Test Guidelines, [email protected], +(33-1) 45 24 98 44 This document, as well as any data and map included herein, are without prejudice to the status of or sovereignty over any territory, to the delimitation of international frontiers and boundaries and to the name of any territory, city or area. 2 │ Foreword This Adverse Outcome Pathway (AOP) on Aryl hydrocarbon receptor activation leading to uroporphyria, has been developed under the auspices of the OECD AOP Development Programme, overseen by the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST), which is an advisory group under the Working Group of the National Coordinators for the Test Guidelines Programme (WNT). The AOP has been reviewed internally by the EAGMST, externally by experts nominated by the WNT, and has been endorsed by the WNT and the Working Party on Hazard Assessment (WPHA) in xxxxx. Through endorsement of this AOP, the WNT and the WPHA express confidence in the scientific review process that the AOP has undergone and accept the recommendation of the EAGMST that the AOP be disseminated publicly.
    [Show full text]
  • On Tuning the Fluorescence Emission of Porphyrin Free Bases Bonded to the Pore Walls of Organo-Modified Silica
    Molecules 2014, 19, 2261-2285; doi:10.3390/molecules19022261 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article On Tuning the Fluorescence Emission of Porphyrin Free Bases Bonded to the Pore Walls of Organo-Modified Silica Rosa I. Y. Quiroz-Segoviano 1, Iris N. Serratos 1, Fernando Rojas-González 1, Salvador R. Tello-Solís 1, Rebeca Sosa-Fonseca 2, Obdulia Medina-Juárez 1, Carmina Menchaca-Campos 3 and Miguel A. García-Sánchez 1,* 1 Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco 186, Vicentina, D. F. 09340, Mexico 2 Departamento de Física, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco 186, Vicentina, D. F. 09340, Mexico 3 Centro de Investigación en Ingeniería y Ciencias Aplicadas, UAEM, Av. Universidad 1001, Col. Chamilpa, C.P. 62209, Cuernavaca Mor., Mexico * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +52-55-5804-4677; Fax: +52-55-5804-4666. Received: 24 December 2013; in revised form: 29 January 2014 / Accepted: 7 February 2014 / Published: 21 February 2014 Abstract: A sol-gel methodology has been duly developed in order to perform a controlled covalent coupling of tetrapyrrole macrocycles (e.g., porphyrins, phthalocyanines, naphthalocyanines, chlorophyll, etc.) to the pores of metal oxide networks. The resulting absorption and emission spectra intensities in the UV-VIS-NIR range have been found to depend on the polarity existing inside the pores of the network; in turn, this polarization can be tuned through the attachment of organic substituents to the tetrapyrrrole macrocycles before bonding them to the pore network.
    [Show full text]
  • 1 INTRODUCTION Corroles Are Tetrapyrrolic Molecules, Maintaining
    1 Chapter 1 INTRODUCTION Corroles are tetrapyrrolic molecules, maintaining the skeletal structure of corrin, with its three meso carbon positions and one direct pyrrole-pyrrole linkage, and possessing the aromaticity of porphyrins (see figure 1.1). First reported by Johnson and Kay in 1965[1], corroles were the end product of a many step synthetic scheme, finally being formed by the photocyclization of a,c-biladienes. While this last step was simple, with 20-60% yields, the route to a,c-biladienes was far from easy. Indeed, the overall reaction from readily available starting materials to corrole was a multi-step synthesis, with poor yields in many of the reactions. Thus, while corroles have been known for more than 40 years, research in the field was slow to progress. It wasn’t until the discovery of new synthetic methods for corroles developed in 1999 by different groups working independently that research in this area really started to expand[2]. This is not to say, however, that the field of corrole research was non-existent prior to the development of the new methodologies. While it was slow growing after the initial publication, it was far from stagnant, and many of the interesting properties of corroles were investigated by different groups. Among these properties is their ability to stabilize unusually high formal oxidation states of metal ions, such as iron(IV), cobalt(IV), and cobalt(V)[3, 4]. In fact, one particular corrole available through a method developed by Zeev 2 N N H N N H H N N N N a b N N H H H N N c Figure 1.1.
    [Show full text]
  • ABSTRACT ZHANG, SHAOFEI. Synthesis Of
    ABSTRACT ZHANG, SHAOFEI. Synthesis of Bacteriochlorins and the Full Skeleton of Bacteriochlorophylls. (Under the direction of Professor Jonathan S. Lindsey.) Bacteriochlorins, the core macrocycle ring of natural bacteriochlorophylls, are characterized by their ability to absorb near infrared light (700 – 900 nm), which makes them attractive candidates in variety of photophysical studies and applications. The previously established de novo synthesis, which relies on the acid-catalyzed self-condensation of dihydrodipyrrin–acetal, provides access towards diverse bacteriochlorins, but has its limitations. This dissertation describes the development of new strategies for bacteriochlorin synthesis. Firstly, a route towards previously unknown tetra-alkyl bacteriochlorins (e.g., alkyl = Me, or –CH2CO2Me) is established (Ch. 2). Secondly, explorations of synthetic approaches to unsymmetrically substituted bacteriochlorins through electrocyclic reactions of linear tetrapyrrole intermediates are described. Four new unsymmetrically substituted bacteriochlorins and one new tetradehydrocorrin were produced, albeit in low yields (Ch. 3). Thirdly, a new method to construct bacteriochlorin macrocycle with concomitant Nazarov cyclization to form the annulated isocyclic ring, is established. Five new bacteriochlorins, which are closely anlogues of bacteriochlorophyll a, bearing various substituents (alkyl/alkyl, aryl, and alkyl/ester) at positions 2 and 3 and 132 carboalkoxy groups (R = Me or Et) were constructed in 37−61% yield from the bilin analogues
    [Show full text]
  • Cheminformatics for Genome-Scale Metabolic Reconstructions
    CHEMINFORMATICS FOR GENOME-SCALE METABOLIC RECONSTRUCTIONS John W. May European Molecular Biology Laboratory European Bioinformatics Institute University of Cambridge Homerton College A thesis submitted for the degree of Doctor of Philosophy June 2014 Declaration This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. This dissertation is not substantially the same as any I have submitted for a degree, diploma or other qualification at any other university, and no part has already been, or is currently being submitted for any degree, diploma or other qualification. This dissertation does not exceed the specified length limit of 60,000 words as defined by the Biology Degree Committee. This dissertation has been typeset using LATEX in 11 pt Palatino, one and half spaced, according to the specifications defined by the Board of Graduate Studies and the Biology Degree Committee. June 2014 John W. May to Róisín Acknowledgements This work was carried out in the Cheminformatics and Metabolism Group at the European Bioinformatics Institute (EMBL-EBI). The project was fund- ed by Unilever, the Biotechnology and Biological Sciences Research Coun- cil [BB/I532153/1], and the European Molecular Biology Laboratory. I would like to thank my supervisor, Christoph Steinbeck for his guidance and providing intellectual freedom. I am also thankful to each member of my thesis advisory committee: Gordon James, Julio Saez-Rodriguez, Kiran Patil, and Gos Micklem who gave their time, advice, and guidance. I am thankful to all members of the Cheminformatics and Metabolism Group.
    [Show full text]
  • A Primitive Pathway of Porphyrin Biosynthesis and Enzymology in Desulfovibrio Vulgaris
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4853–4858, April 1998 Biochemistry A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris TETSUO ISHIDA*, LING YU*, HIDEO AKUTSU†,KIYOSHI OZAWA†,SHOSUKE KAWANISHI‡,AKIRA SETO§, i TOSHIRO INUBUSHI¶, AND SEIYO SANO* Departments of *Biochemistry and §Microbiology and ¶Division of Biophysics, Molecular Neurobiology Research Center, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21, Japan; †Department of Bioengineering, Faculty of Engineering, Yokohama National University, 156 Tokiwadai, Hodogaya-ku, Yokohama 240, Japan; and ‡Department of Public Health, Graduate School of Medicine, Kyoto University, Sakyou-ku, Kyoto 606, Japan Communicated by Rudi Schmid, University of California, San Francisco, CA, February 23, 1998 (received for review March 15, 1998) ABSTRACT Culture of Desulfovibrio vulgaris in a medium billion years ago (3). Therefore, it is important to establish the supplemented with 5-aminolevulinic acid and L-methionine- biosynthetic pathway of porphyrins in D. vulgaris, not only methyl-d3 resulted in the formation of porphyrins (sirohydro- from the biochemical point of view, but also from the view- chlorin, coproporphyrin III, and protoporphyrin IX) in which point of molecular evolution. In this paper, we describe a the methyl groups at the C-2 and C-7 positions were deuter- sequence of intermediates in the conversion of uroporphy- ated. A previously unknown hexacarboxylic acid was also rinogen III to coproporphyrinogen III and their stepwise isolated, and its structure was determined to be 12,18- enzymic conversion. didecarboxysirohydrochlorin by mass spectrometry and 1H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzy- MATERIALS AND METHODS matic steps: (i) methylation of the C-2 and C-7 positions of Materials.
    [Show full text]
  • Biosynthesis of Vitamin B12: Concerning the Origin of the Methine Protons of the Corrin Nucleus (Deuterium Isotope Effects/'3C NMR Spectroscopy) A
    Proc. Nati. Acad. Sci. USA Vol. 84, pp. 6616-6618, October 1987 Chemistry Biosynthesis of vitamin B12: Concerning the origin of the methine protons of the corrin nucleus (deuterium isotope effects/'3C NMR spectroscopy) A. IAN SCOTT, MASAHIRO KAJIWARA, AND PATRICIO J. SANTANDER Center for Biological NMR, Department of Chemistry, Texas A&M University, College Station, TX 77843 Communicated by D. H. R. Barton, June 8, 1987 ABSTRACT 13C NMR spectroscopy has been used to C-19 in cobester as discussed above (6). It is also of interest locate six deuterium atoms incorporated biosynthetically on the to note that there is no deuterium at C-10, a position known periphery of the corrin nucleus of vitamin B12 (cyanocobala- to undergo prototropic exchange under acidic conditions (9). min) derived from cells of Propionibacterium shermanii grown Confirmation and extension of the assignments were made in a medium containing 50% 21120 and 13C-enriched 6- by analysis of the spectrum of cyanocobalamin (1) obtained aminolevulinic acid. The implications of these results for the by the same procedure but in the presence of [3-13C]ALA, mechanism of vitamin B12 biosynthesis are discussed, and it is which labels a different set of carbon centers (see Fig. 3). concluded that the same oxidation level of the intermediates is Thus, in addition to an upfield a shift on C-18, p 2H shifts are maintained throughout the biosynthetic pathway, from 8- found at C-2, C-3a, C-7, C-8a, C12, C-13a, and C-18 (Table 2). aminolevulinic acid to corrin. In addition, the C12 P-methyl group has a single deuteron substituent as discerned in the double shift on C12 (from 2H Our knowledge ofthe carbon balance ofthe pathway leading to at C-13 and C12 C1H22H).
    [Show full text]
  • Preparation of Tetrapyrrole-Amino Acid Covalent Complexes
    I'lunt I'ht.siol.Ritx ltt'nt. 1996. -14 (3). 393-39lt Preparation of tetrapyrrole-amino acid covalent complexes Leszek Fiedorl'2*, Varda Rosenbach-Belkinl, Maruthi Sail and Avigdor Scherzl I BiochernistryDepartment. The Weizn-rannInstitute of Science.76100 Rehovot.Israel. I Prcscntaddress: Institute of Molecular BiologSr.Ja-ciellonian University. Al. Mickiewicza 3. 3 l- 120 Cracow. Poland. ':'Author to whom correspondenceshould bc addrcsscd(fax +48-12-336907:E-mail fiedor@)mol.uj.edu.pl) Abstract The presentedsynthetic approach towards chcn'rical modifications of chlorophylls(Chls) provides a perspectivcto construct model systems. where tetrapyrrole-aminoacid and tetrapyrrole-peptideinteractions coulcl be studied in covalent rnodel compor,rncls. The approach relies on thc lact that in Chls the | 7r propionic rcid sidc chain docs not participatc in the tetrapl'rroleii--electron system. It makes use of a plant enzvmechlolophyllase (EC 3.1.1.1,+).which lrr lilo and in yitrc catalysesreactions at this sidc function. The transesterilicationand hyclrolysisenzymatic rerctions are useful on a preparativescale. ln the transesterificationreaction. a desiredamino acid rcsiduc posscssirrgprimary hydloxyl group can be directly attachedto the propiorric acid side chain o1' Chl. This mcthod allows to replace the phytyl moiety in Chls n'ith seline. The r:rtherreaction. enzyrratic hydrolysis of Chls, yields chlorophyllides and opens a convenientroutc fbr furthcr rnodifications.If sufliciently mild synthetic mcthodsarc uscd. such as catalysisw,ith ,l-dimethyl arnino pyridine or activationwith N-hvdroxvsuccinimide.an arrino acid or peptide residuecan be covalentlybound to chlorophyllides' carboxylic group. lear,'ingthe essentialclectlonic structure of Chl intact. The activation w'ith N-hydroxvsuccininridcallows fbr the coupling cvrn in aqueous rncdia.
    [Show full text]
  • Catabolism of Tetrapyrroles As the Final Product of Heme Catabolism (Cf Scheme 1)
    CHEMIE IN FREIBURG/CHIMIE A FRIBOURG 352 CHIMIA 48 (199~) Nr. 9 (Scl'lcmhcr) ns itu Chimia 48 (/994) 352-36/ heme (1), at the a-methene bridge (C(5)) €> Neue Sclnveizerische Chemische Gesellschaft producing CO and an unstable Felli com- /SSN 0009-4293 plex. The latter loses the metal ion to yield the green pigment protobiliverdin IXa (usually abbreviated to biliverdin (2)), which is excreted by birds and amphibia, Catabolism of Tetrapyrroles as the final product of heme catabolism (cf Scheme 1). The iron is recovered in the protein called ferritin and can be reutilized Albert Gossauer* for the biosynthesis of new heme mole- cules. As biliverdin (2) has been recog- nized to be a precursor in the biosynthesis of phycobilins [9], a similar pathway is Abstract. The enzymatic degradation of naturally occurring tetrapyrrolic pigments probably followed for the biosynthesis of (heme, chlorophylls, and vitamin B 12) is shortly reviewed. this class oflight-harvesting chromophores 1. Introduction pounds known so far are synthesized, have Scheme I. Catabolism (!{ Heme ill Mammals been already elucidated, it may be antici- In contrast to the enormous amount of pated that the study of catabolic processes work accomplished by chemists in the will attract the interest of more chemists elucidation of biosynthetic pathways of and biochemists in the near future. secondary metabolites (terpenes, steroids, alkaloids, among others), only a few at- tempts have been made until now to un- 2. Heme Catabolism derstand the mechanisms oftheirdegrada- tion in living organisms. A possible rea- It has been known for over half a cen- son for this fact is the irrational association tury that heme, the oxygen-carrier mole- of degradation (catabolism: greek Kara= cule associated with the blood pigment down) with decay and, thus, with unattrac- hemoglobin, is converted in animal cells tive dirty colors and unpleasant odors.
    [Show full text]