Charakterisierung der Vinorin-Synthase aus Rauvolfia serpentina durch Reinigung, Expression, Mutation und Kristallisation

ABSTRACTDie vorliegende Arbeit befasste sich mit der Reinigung,heterologen Expression, Charakterisierung, molekularenAnalyse, Mutation und Kristallisation des EnzymsVinorin-Synthase. Das Enzym spielt eine wichtige Rolle inder Ajmalin-Biosynthese, da es in einerAcetyl-CoA-abhängigen Reaktion die Umwandlung desSarpagan-Alkaloids 16-epi-Vellosimin zu Vinorin unterBildung des Ajmalan-Grundgerüstes katalysiert. Nach der Reinigung der Vinorin-Synthase ausHybrid-Zellkulturen von Rauvolfia serpentina/Rhazya strictamit den fünf chromatographischen TrennmethodenAnionenaustauschchromatographie an SOURCE 30Q, HydrophobeInteraktionen Chromatographie an SOURCE 15PHE,Chromatographie an MacroPrep Ceramic Hydroxyapatit,Anionenaustauschchromatographie an Mono Q undGrößenausschlußchromatographie an Superdex 75 konnte dieVinorin-Synthase aus 2 kg Zellkulturgewebe 991fachangereichert werden.Das nach der Reinigung angefertigte SDS-Gel ermöglichte eineklare Zuordnung der Protein-Bande als Vinorin-Synthase.Der Verdau der Enzymbande mit der Endoproteinase LysC unddie darauffolgende Sequenzierung der Spaltpeptide führte zuvier Peptidsequenzen. Der Datenbankvergleich (SwissProt)zeigte keinerlei Homologien zu Sequenzen bekannterPflanzenenzyme. Mit degenerierten Primern, abgeleitet voneinem der erhaltenen Peptidfragmente und einer konserviertenRegion bekannter Acetyltransferasen gelang es, ein erstescDNA-Fragment der Vinorin-Synthase zu amplifizieren. Mit derMethode der RACE-PCR wurde die Nukleoidsequenzvervollständigt, was zu einem cDNA-Vollängenklon mit einerGröße von 1263 bp führte, der für ein Protein mit 421Aminosäuren (46 kDa) codiert.Das Vinorin-Synthase-Gen wurde in den pQE2-Expressionsvektorligiert, der für einen N-terminalen 6-fachen His-tagcodiert. Anschließend wurde sie erstmals erfolgreich in E.coli im mg-Maßstab exprimiert und bis zur Homogenitätgereinigt. Durch die erfolgreiche Überexpression konnte dieVinorin-Synthase eingehend charakterisiert werden. DerKM-Wert für das Substrat Gardneral wurde mit 20 µM, bzw.41.2 µM bestimmt und Vmax betrug 1 pkat, bzw. 1.71 pkat.Nach erfolgreicher Abspaltung des His-tags wurden diekinetischen Parameter erneut bestimmt (KM- Wert 7.5 µM, bzw.27.52 µM, Vmax 0.7 pkat, bzw. 1.21 pkat). Das Co-Substratzeigt einen KM- Wert von 60.5 µM (Vmax 0.6 pkat). DieVinorin-Synthase besitzt ein Temperatur-Optimum von 35 °Cund ein pH-Optimum bei 7.8.Homologievergleiche mit anderen Enzymen zeigten, dass dieVinorin-Synthase zu einer noch kleinen Familie von bisher 10Acetyltransferasen gehört. Alle Enzyme der Familie haben einHxxxD und ein DFGWG-Motiv zu 100 % konserviert. Basierendauf diesen Homologievergleichen und Inhibitorstudien wurden11 in dieser Proteinfamilie konservierte Aminosäuren gegenAlanin ausgetauscht, um so die Aminosäuren einer in derLiteratur postulierten katalytischen Triade(Ser/Cys-His-Asp) zu identifizieren.Die Mutation aller vorhandenen konservierten Serine undCysteine resultierte in keiner Mutante, die zumvollständigen Aktivitätsverlust des Enzyms führte. Nur dieMutationen H160A und D164A resultierten in einemvollständigen Aktivitätsverlust des Enzyms. Dieses Ergebniswiderlegt die Theorie einer katalytischen Triade und zeigte,dass die Aminosäuren H160A und D164A exklusiv an derkatalytischen Reaktion beteiligt sind.Zur Überprüfung dieser Ergebnisse und zur vollständigenAufklärung des Reaktionsmechanismus wurde dieVinorin-Synthase kristallisiert. Die bis jetzt erhaltenenKristalle (Kristallgröße in µm x: 150, y: 200, z: 200)gehören der Raumgruppe P212121 (orthorhombisch primitiv) anund beugen bis 3.3 Å. Da es bis jetzt keine Kristallstruktureines zur Vinorin-Synthase homologen Proteins gibt, konntedie Struktur noch nicht vollständig aufgeklärt werden. ZurLösung des Phasenproblems wird mit der Methode der multiplenanomalen Dispersion (MAD) jetzt versucht, die ersteKristallstruktur in dieser Enzymfamilie aufzuklären.ABSTRACTThe Indian medicinal plant Rauvolfia serpentina (L.) Benthamex Kurz is a well known source for the isolation of theantiarrhythmic Ajmaline, a monoterpenoid indole alkaloidapplied in the treatment of heart disorders for manydecades. The biosynthesis of this structural complex indolealkaloid bearing nine chiral carbons has been wellelaborated at the enzymatic level during recent years. Thebiosynthesis of Ajmaline is catalysed by about 10 differentenzymes. Purification of the enzymes is difficult because ofthe very limited occurrence in plant derived Rauvolfiasystems (hairy roots, tissue or cell cultures). Thereforeheterologous expression of the proteins in bacteria or yeastcells is necessary to study the involved enzyme mechanismsin more detail and to understand the complicatedbiosynthetic formation of this drug. One enzyme playing a very important role in the Ajmalinebiosynthesis is the Vinorine Synthase (VS). It converts thesarpagine-type alkaloid epi-Vellosimine to the ajmaline-typeVinorine by an Acetyl-CoA dependent reaction Results:After purification of vinorine synthase fromRauvolfia/Rhazya cell suspension cultures, the amino acidsequence of 4 endoproteinase Lys-C generated peptides wasdetermined. The sequence and position of these internalpeptides are indicated by grey boxes in Fig. 3. A comparisonof these amino acid sequences with those available in theGenBankTM/EMBL sequence data bases indicated no similarityto any known protein. From these 4 peptide-fragmentsdegenerated primer pairs were designed in order to generatethe first cDNA fragment of vinorine synthase by PCR. Withthe 12 possible primer combinations it was not possible tocreate a relevant cDNA fragment.Sequence-alignment studies of acetyltransferases from otherplants revealed a 100% conserved region near the carboxylterminus (Grothe, Lenz et al. 2001). With a degeneratedantisense primer derived from this conserved region(DFGWG-motiv) in combination with a sense primer based uponan internal peptide sequence of vinorine synthase (FVFDKEK)we obtained a fragment of 500 basepairs with an ORF showingidentities of up to 32 % to the acetyltransferases.The positions of these peptides are indicated by arrows inFig. 3. RACE- PCR was then used to generate the 5`- and3`-ends of the putative vinorine synthase cDNA. Thetranslation of the complete vinorine synthase cDNA yielded apolypeptide of 423 amino acids (46 kDa).The vinorine synthase cDNA was constructed to express therecombinant protein with an N-terminal six times histidinetag and could therefore be purified by nickel-affinitychromatography. Like this vinorine synthase was expressed inE.Coli for the first time and purified to homogeneityleading to mg amounts of highly purifie enzyme (2mg/l). Theactivity of the pure recombinant enzyme was tested with astandard incubation as described in experimental proceduresand the result of the incubation was tested by HPLC. Theidentity of the enzymatic reaction product methoxyvinorinewas also proved by MS analysis .Vinorine synthase is very substrate specific. Next to it`snatural substrate 16 epi-vellosimine only the11-methoxyderivative Gardneral is accepted.16-epivellosimine epimerises within 20 minutes and is thennot accepted as a substrate anymore. Gardneral has a lessertendency of epimerization and is therefore used as asubstrate in all following experiments.The apparent KM value for Garneral was determined to be 27µMolar at a fixed concentration of acetyl-CoA of 725 µM. Theapparent KM value for acetyl-CoA was determined to be 60.5µM at a fixed concentration of Gardneral of 35.7 µM. TheVmax for the acetylation of Gardneral was 1.21 pkat with atemprature optimum of 35 °C and a pH of 7.8 under standardassay conditions.For further characterization a series of inhibitionexperiments were performed. Several known inhibitorsreacting selectively with the amino acids serine, histidineand cysteine were tested on the enzyme. AEBSFa selectiveserine modifying agent, inhibited the enzyme activity 100 %.The selective cysteine inhibitor E-64 reduced enzymeactivity to 50 %. With the serine-cysteine inhibitors PMSF,TPCK, TLCK inhibition from 100-58 % was observed. Inhibitionwith the unselective cysteine-inhibitor Hg2+ and with theunselective histidine inhibitor DEPC lead in both cases to acomplete loss of activity. These experiments show that theamino acids serine, cysteine and histidine might play a rolein the catalytic reactionSequence alignment studies showed that vinorine synthasebelongs to a small family of acetyltransferases. The familyconsists of 10 members so far, all of them of plant origin.The majority of the enzymes belong to pharmaceuticallyimportant plants like papaver. Histidine and aspartatresidues (HxxxD) are highly conserved as well as a DFGWGmotif near the C-terminus of the proteins. The conservedhistidines and aspartates are thought to be involved incatalysisi as characterized by x-ray crystallography for thebacterial enzymes chloramphenicol acetyltransferase anddihydrolipoamide acetyltransferase. Based on these alignemtstudies the aminoacids serine, cysteine and histidine weresuggested to be involved in the catalytic reaction. Withthese suggestions and the previous inhibitor studies somesite-directed mutagenesis experiments were performed to getmore information about the catalytic domains of the enzymeThe conserved amino acids serine 29, cysteine 89, cysteine149, histidine 160, asparagine 293 and serine 413 wereexchanged against alanin. After expression and purificationthe KM values were determined. Mutation H160A resulted in acomplete loss of activity. Therefore it was not possible todetermine the KM value.The mutants S29A (KM 4.65 µM) and C149A (KM 1.06 µM) showeda great loss of activity . Interestingly they appear to havesmaller KM values than the wildtype. C149A, C89A and Asn293Ashowed no significant loss of activity, therefore the KMvalues are similar to the wildtype.To identify more members of a putative catalytic triade allthe remaining conserved serines and aspartates were mutatedagainst alanin. Due to a shortage of substrate we onlydetermined the activity of the mutants and not the KMvalues. Mutation D164A resulted in a complete loss ofactivity. Mutations S243A, D32A and D361A lead to asignificant decrease of activity whereas the mutations S16A,S68A, D359A showed no difference to the wildtype. Table 5shows the specific activity of all 13 mutations.For further elucidation of the catalytic mechanism ofvinorine synthase crystallization studies with thehanging-drop-method were performed. The 6x His tag wascleaved off and 160 different precipitation-buffers weretried out. For crystallisation experiments protein solutionsof 1-5 mg/ml were used. The best results were finallyobtained under the given conditions (2mg/ml protein, 0.1MHepes pH 7.3, 1.5 % PEG 400, 2 M ammonium sulfate). With700 µl reservoir solution and droplets consisting of 3 µlprotein and 3 µl crysatllization reagentcrystals appearedwithin 4-5 d. The crystals grew to to maximum dimensions of150 x 200 x 200 µm. Prior to X-ray data collection, thecrystals were transferred to a cryoprotectant bufferconsisting of reservoir solution supplemented with 20 %glycerol.Data were collected at the EMBL-Outstation Hamburg, beamlineBW7B using a wavelength of 0.84570 Å. Data were processed upto a resolution of 3.3 Å. Based on the data set the spacegroup was determined to be P212121 with the unit cellparameters a=079.6 Å, b=89.7 Å, c=135.3 Å.Since there are no protein structure available that showsignificant sequence homology with vinorine synthase fromRauvolfia, recombinant expression will be used to obtain aselenomethionine-modified protein. The structure will bedetermined using the multiple-wavelenght anomalousdispersion (MAD) approach