Hyperforin biosynthesis [Elektronische Ressource] : characterization and purification of a prenyltransferase from Hypericum calycinum cell cultures / von Zakia Boubakir

Hussein Kassem

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2006
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Hyperforin biosynthesis  Characterization and purification of a prenyltransferase fromHypericum calycinumcell cultures Von der Fakultät für Lebenswissenschaften der Technischen Universität CaroloWilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat) genehmigte D i s s e r t a t i o n von Zakia Boubakir aus Derna, Libyen

1. Referee: Prof. Dr. Ludger Beerhues 2. Referee: PD Dr. Dietrich Ober eingereicht am: 22.02.2006

mündliche Prüfung (Disputation) am: 25.04.2006

Publications Parts of this work have previously been published with permission of the Faculty of Chemistry and Pharmacy, represented by the mentor of this work: Publications Boubakir, Z., Beuerle,T., Liu, B., Beerhues, L. (2005). The first prenylation step in hyperforin biosynthesis. Phytochemistry 66: 5157 Klingauf, P., Beuerle, T., Mellenthin, A., ElMoghazy, S.A.M., Boubakir, Z., Beerhues, L. (2005). Biosynthesis of the hyperforin skeleton inHypericum calyciumcell cultures. Phytochemistry 66 : 139145 Beerhues, L., Abd ElMawla, A., Beuerle, T., Boubakir, Z., Klingauf, P., Liu, B. (2005) Biosynthesis of active plant polyketide derivatives. Proceedings of the 9th International Congress "Phytopharm 2005" and the Young Scientists Meeting of the Phytochemical Society of Europe on "Plants and Health" (Makarov, V.G., ed.) PP 3946 Charchoglyan, A., Abrahamyan, A., Boubakir, Z., Gulder, T., Fujii, I., Bringmann, G., Vardapetyan, H., Kutchan, T.M., Ebizuka, Y., Beerhues, L. Hyperforin and secohyperforin suggest a branch point in the biosynthetic pathway. In preparation. Presentations Short lecture Boubakir, Z., Liu, B., Zapletalová, P., Beerhues, L. (2003) Molecular analysis of the biosynthesis of the active compounds inHypericum perforatum. DPhGJahrestagung (Würzburg, 08.11. Oktober 2003) Boubakir, Z., Beerhues, L. (2005) Purification of a prenyltransferase related to hyperforin biosynthesis KurtMothesDoktorandenWorkshop Sekundärstoffwechsel (Halle, 05.07. Oktober 2005) Poster Boubakir, Z., Beuerle, T., Beerhues, L. (2004) Prenylation reactions in hyperforin biosynthesis Botanikertagung der Deutschen Botanischen Gesellschaft und Vereinigung für Angewandte Botanik (Braunschweig, 05.10. September 2004)

ACKNOWLEDGMENT I would like to express my special gratitude to my supervisor Professor Dr. Ludger Beerhues for introducing me to this project and for providing excellent scientific facilities and friendly working conditions. He kindly supported me, and he always had time for questions and discussions. I am grateful to Professor Dr. Dietrich Ober for his interest in this work and for taking over the coreferee. I am also thankful to Prof. Dr. Benye Liu for his ideas and valuable advices throughout my PhD work, Dr. Till Beuerle for his help with chemical synthesis and analysis of reference and enzymatic compounds, Dr. Rainer Lindigkeit for many helpful advices with software problems and his computer assistance at any time. Special thanks go to all staff members of the Institute of Pharmaceutical Biology of TU Braunschweig, present or past, for a friendly and synergistic cooperation, to Mrs. Ines Rahaus for providing a stimulating and fun environment and for all her help. My thanks also go to Mrs. Hoda Mohagheghi, for her help with 2D electrophoresis. I am grateful to the Libyan government for granting me a PhD scholarship. Especially, I would like to give my deep thanks to my husband Hussein for his patient love. Without his support and love, I would have not been ableto complete this work. I also thank my kids Retaj and Roba for their patience and emotional support. Last, but not least, my sincere thanks are to my family: my parents for educating me with aspects from both arts and sciences and for unconditional support and encouragement to finish my PhD work; my brothers and my sisters for their everloving care towards me throughout.

Contents I Contents List of Tables...............................................................................................................................V List of Figures............................................................................................................................VΙ Abbreviations...............................................................................................................................X

1. Introduction..........................................................................................................................1

 1.1. St. John’s wort extracts………………………………………...………………………...1  1.2. Depression........………………………………………………………………………......2  1.3.Hypericum perforatumL ………………….………………………….…........................3  1.3.1 Constituents...............................................................................................................4  1.4. Hyperforin......….……….…………………………………………………….……….....6  1.4.1 Pharmacology............................................................................................................8  1.5. Prenyltransferase.……………………………….…………………………………...…...9  1.6.Hypericum calycinumL..….………..………………………………………..…………10  1.7. Research strategies and objectives……...………………………………………………11

2. Materials..............................................................................................................................12

nutrient media, solutions, buffers, equipment....….……….…….…….......122.1. Chemicals, 2.1.1 Chemicals…...………………………………………….…………………….…..12 2.1.2 Nutrient media for plant tissue cultures....……………......……………………..14 2.1.3 Solutions, buffers for biochemical analysis...........................................................16 2.1.4 Solutions, buffers for protein purification……........…...…..…………................17 2.1.5 Buffers and solutions for gel electrophoresis.………....…..……………………..18 2.1.6 Buffers for firstdimension isoelectric focusing (IEF)….........…………………..19 2.1.7 Equipment........….……..………..…………………...…....……………............. 20  2.2. Plant material......……………..….......……………………..…………………............21 2.1.1 Cultivation of callus and suspension cultures….......................……….........……21  2.1.2 Cell harvest….............…..………..........……………............................................21 3. Methods...............................................................................................................................22

 3.1. Syntheses and analytical work.........................................................................................22 3.1.1 Synthesis of dimethylallyl diphosphate…………...................................................22 3.1.1.1 Purification of synthetic dimethylallyl diphosphate....................................23 3.1.1.2 Thin layer chromatography (TLC)..............................................................23

Contents II  3.1.1.3 NMRanalysis.............................................................................................23  3.1.2 Synthesis of dimethylallylphlorisobutyrophenone............................................24 3.1.2.1 Purification of synthetic dimethylallylphlorisobutyrophenone……...........24 3.1.2.2 Thin layer chromatography (TLC)..............................................................24 3.1.2.3 High performance liquid chromatography.................................................25 3.1.2.3.1 Instrumental.........................................................................................25 3.1.2.3.2 Mobile phases......................................................................................25 3.1.2.4 GCMS analysis..........................................................................................26 3.1.2.4.1 Instrumental .......................................................................................26 3.1.2.4.2 Sample preparation.............................................................................26 3.1.3 Synthesis of dimethylallylphloracetophenone…...............................................26 3.1.3.1 Purification of synthetic dimethylallylphloracetophenone…......................27 3.1.3.2 Thin layer chromatography (TLC)..............................................................27  3.2. Enzyme extraction and incubation.................................................................................27 3.2.1 Preparation of cellfree extracts...........................................................................27 3.2.2 Protein determination...........................................................................................27 3.2.3 Enzyme assay.......................................................................................................28 3.2.4 Enzyme preparation for ultracentrifugation.........................................................28 3.2.5 Linearity with protein amount and incubation time.............................................28  3.3. Characterization of the prenyltransferase…..................................................................29 3.3.1 Determination of pH and temperature optima.....................................................29 3.3.2 Study of substrate specificity…...........................................................................29 3.3.3 Cofactor requirement...........................................................................................29 3.3.4 Determination of kinetic data..............................................................................29  3.4. Purification of the prenyltransferase.............................................................................30 3.4.1.1 Ammonium sulphate precipitation...............................................................30 3.4.1.2 DEAEanion exchange chromatography…..………………………………30 3.4.1.2.1 DEAEcolumn with 1 ml gel volume......................................................30 3.4.1.2.1 DEAEcolumn with 5 ml gel volume......................................................32  3.4.1.3 Hydroxylapatiteadsorption chromatography................................................33 3.4.1.4 Hydrophobic interaction chromatography.....................................................33 3.4.1.5 MonoQ anion exchange chromatography................................................... 34 3.4.1.6 Gel filtration...................................................................................................34  3.5.2 Purification of the prenyltransferase to apparent homogeneity..........................36

Contents III  3.5. SDS Polyacrylamide Gel Electrophoresis (SDSPAGE).............................................36 3.5.1 Preparation of gel and samples.............................................................................36 3.5.2 Electrophoresis and detection of the protein bands..............................................37  3.6. Twodimensional (2D) electrophoresis........................................................................38 3.6.1 Sample preparation………………………………..............................………….39 3.6.2 FirstDimension Isoelectric Focusing (IEF) ........................................................39 3.6.3 Seconddimension SDS PAGE…….....................................................................41 3.6.4 Detection and analysis of 2D protein spots.........................................................42

4. Results................................................................................................................................43

4.1. Detection of prenyltransferase activity........................................................................43 4.1.1 Synthesis of the reference dimethyallylphlorisobutyrophenone........................... 45 4.1.2 Analysis of the enzymatic product dimethyallylphlorisobutyrophenone..............47 4.2. Characterization of prenyltransferase…...................................................................... 48 2.2.1Linearity with protein amount and incubation time..............................................48 2.2.2Determination of pH and temperature optima......................................................49 2.2.3Cofactor requirement............................................................................................50 2.2.4Effect of ascorbic acid..........................................................................................50 2.2.5Substrate specificity...............….......................................................................... 52 2.2.6Synthesis of the reference dimethyallylphloracetophenone.................................52 2.2.7Analysis of the enzymatic product dimethyallyl phloracetophenone...................55 2.2.8Determination of kinetic data...............................................................................57 2.2.9Prenyltransferase activity in cell fractions after ultracentrifugation....................59  2.2.10 Changes in prenyltransferase activity and adhyperforin formation during cell  culture growth.....................................................................................................59 4.3. Purification of the prenyltransferase...........................................................................60 4.3.1 Fractionation by ammonium sulphate precipitation.............................................60 4.3.2 Column chromatography ....................................................................................61 4.3.2.1 DEAEanion exchange chromatography ......................................................61 4.3.2.1.1 DEAEcolumn with 1 ml gel volume...................................................61 4.3.2.1.2 DEAEcolumn with 5 ml gel volume...................................................63 4.3.2.2 Hydroxylapatiteadsorption chromatography..............................................63 4.3.2.3 Hydrophobic interaction chromatography...................................................65 4.3.2.4 Mono Q anion exchange chromatography................................................66

Contents IV 4.3.2.5 Gel filtration................................................................................................. 68  4.3.3 Purification of the prenyltransferase ..................................................................70 4.3.4 SDS polyacrylamide gel electrophoresis (SDSPAGE)..................................... 73 4.3.4.1 Preparation of samples................................................................................73 4.3.4.2 Electrophoresis and detection of the protein bands.....................................73  4.3.5 Determination of the molecular mass..................................................................75 4.3.5.1 Determination of the subunit molecular mass by SDSPAGE....................75 4.3.5.2 Determination of the native molecular mass by gel filtration.....................75  4.3.6 Twodimensional (2D) electrophoresis..............……………………...........….76 4.3.6.1 FirstDimension Isoelectric Focusing (IEF)……….....….………………76 4.3.6.2 Seconddimension SDS PAGE………………………….………………..76 4.3.6.3 Detection and analysis of 2D resolved protein spots…........….…………76 4.3.7 Partial sequencing of the separated polypeptides………..........…….………….77 5. Discussion...........................................................................................................................79 6. Summary............................................................................................................................94 7. References..........................................................................................................................96 8. Appendices........................................................................................................................108

List of Tables V

List of Tables Tab. 11 Main constituents of St. John's wort.........................................................................6 Tab. 31 Stock solutions and preparation of media...............................................................14 Tab. 32 Buffers for extraction and enzyme incubation........................................................16 Tab. 33 Solution for regeneration of PD10 columns (Amersham Biosciences).................16 Tab. 34 Solution for protein estimation................................................................................16 Tab. 35 Buffers for DEAE and MonoQ anion exchange columns.....................................17 Tab. 36 Buffers for the HAP (CHT5I) column...................................................................17 Tab. 37 Buffers for HIC (Phenyl sepharose high performance column)..............................17 Tab.38 Buffer for Gel Filtration (HiPrep16/60 Sephacryl S200 High Resolution  column)...................................................................................................................17 Tab. 39 Buffers and solutions for gel electrophoresis.........................................................18 Tab. 310 Buffers for FirstDimension Isoelectric Focusing.................................................19 Tab. 311 HPLC program.......................................................................................................24 Tab. 312 HPLC programs.....................................................................................................25 Tab. 313 FPLC program for a 1mlDEAE anion exchange column...................................31 Tab. 314 FPLC program for a 5mlDEAE anion exchange column...................................32 Tab. 315 Recommended concentrations of standard proteins..............................................35 Tab. 316 Silver staining protocol..........................................................................................38 Tab. 317Focusing program..................................................................................................41 Tab. 41 Effect of the amount of protein in the standard assay on the amount of enzyme  product..................................................................................................................48 Tab. 42 Substrate specificity of phlorisobutyrophenone dimethylallyltransferase...........52 Tab. 43 Prenyltransferase activity in cell fractions after ultracentrifugation....................59 Tab. 44 Purification of the prenyltransferase fromH. calycinumcell cultures….............72

List of Figures VI

List of Figures Fig. 11Hypericum perforatumL ...........................................................................................4 Fig. 12 Chemical structure of hyperforins .............................................................................7 Fig. 13Hypericum calycinumL...........................................................................................10 Fig. 14 The first prenylation step in hyperforin biosynthesis...............................................11 Fig. 21 Callus culture of H. calycinum on solid LS medium...............................................21 Fig. 22 Cell suspension culture ofH. calycinumin LS medium..........................................22 Fig. 23 Cell suspension culture ofH. calycinumin BDS medium.......................................22 Fig. 41 HPLC analysis of prenyltransferase assays with phlorisobutyrophenone................43 Fig.42 GCMS analysis of prenyltransferase assays............................................................44 Fig. 43 UV spectrum of synthetic reference dimethylallylphlorisobutyrophenone............45 Fig. 44 Mass spectrum of synthetic reference dimethylallylphlorisobutyrophenone...........46 1 Fig. 45 H NMR spectrum of synthetic reference dimethylallylphlorisobutyrophenone.....46 Fig. 46 Time course of product formation in the prenyltransferase assay...........................48 Fig. 47 pH optimum of the prenyltransferase.......................................................................49 Fig. 48 Temperature optimum of prenyltransferase.............................................................49 Fig. 49 Effect of divalent cations on dimethylallyltransferase activity...............................50 2+ Fig. 410 Dependence of dimethylallyltransferase activity on the concentration of Fe ......51 2+ Fig. 411 Determination of the K m from a LineweaverBurk plot..................51value for Fe Fig. 412 UV spectrum of synthetic reference dimethylallylphloracetophenone.................53 Fig. 413 Mass spectrum of synthetic reference dimethylallylphloracetophenone................53 1 Fig. 414 H NMR spectrum of synthetic reference dimethylallylphloracetophenone...........54 Fig. 415 HPLC analysis of prenyltransferase assays with phloracetophenone.....................55 Fig. 416 GCMS analysis of the enzymatic product dimethylallylphloracetophenone and the  synthetic reference compound (after acetylation)...................................................56 Fig. 417 Dependence of dimethylallyltransferase activity on the concentration of  phlorisobutyrophenone..........................................................................................57 Fig. 418 Determination of the K m  value for phlorisobutyrophenone from a Lineweaver  Burk plot................................................................................................................57 Fig. 419 Dependence of dimethylallyltransferase activity on the concentration of  DMAPP.................................................................................................................58 Fig. 420 Determination of the K m value for DMAPP from a Lineweaver Burk plot..........58

List of Figures VII

Fig. 421 Adhyperforin formation and change in prenyltransferase activity during cell  culture growth………………………………………..............................................60 Fig. 422 Distribution of prenyltransferase activity in ammonium sulphateprecipitated  protein fractions.....................................................................................................61 Fig. 423 Anion exchange chromatography of the protein fraction precipitating between  30 and 65% ammonium sulphate saturation on a 1 mlDEAE Sepharose  column......................................................................................................62 Fig. 424 Detection of prenyltransferase activity in protein fractions eluted from the DEAE  Sepharose column..................................................................................................63 Fig. 425 Hydroxylapatiteadsorption chromatography of the protein fraction precipitating  between 30 and 65% ammonium sulphate saturation on a CHT51 column........64 Fig. 426 Detection of prenyltransferase activity in protein fractions obtained b y  hydroxylapatiteadsorption chromatography.......................................................64 Fig. 427 Hydrophobic interaction chromatography of the protein fraction precipitating  between 30 and 65% ammonium sulphate saturation on a Phenyl Sepharose  column.................................................................................................................65 Fig. 428 Detection of prenyltransferase activity in protein fractions separated on a Phenyl  Sepharose column.................................................................................................66 Fig. 429 Anion exchange chromatography of the protein fraction precipitating between  30 and 65% ammonium sulphate saturation on a MonoQ column.......................67 Fig. 430 Detection of prenyltransferase activity in protein fractions eluted from a Mono  Q column................................................................................................................67 Fig. 431 Elution profile of blue dextran on a Sephacryl S200 HR column........................68 Fig. 432 Elution profile of standard proteins on a Sephacryl S200 HR column.................69 Fig. 433 Calibration curve of standard proteins on Sephacryl S200 HR column...............69 Fig. 434 Diagram depicting the purification scheme applied to the prenyltransferase from H. calycinumcell culture......................................................................................70 Fig. 435 Gel filtration on Sephacryl S200 of the prenyltransferasecontaining protein  fraction obtained by MonoQ anion exchange chromatography..........................72 Fig. 436 SDSPAGE (12% gel) of fractions containing prenyltransferase activity after the  following purification steps. (1) Crude extract; (2) (NH 4 ) 2 SO 4  precipitation; (3) DEAE; (4) HAP; (5)HIC; (6) MonoQ; (7) gel filtration. (M) Molecular mass  markers...................................................................................................................74