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3D-Structure of Vinorine Synthase from Rauvolfia Serpentina in Complex with Its Ligand Acetyl-Coa

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3D-Structure of Vinorine Synthase from Rauvolfia Serpentina in Complex with Its Ligand Acetyl-Coa 3D-Structure of Vinorine Synthase from Rauvolfia serpentina in Complex with its Ligand Acetyl-CoA M. Hill, S. Panjikar1, X. Ma, J. Stöckigt Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudingerweg 5, D-55099 Mainz, Germany 1EMBL Hamburg outstation DESY, Notkestraße 85, D-22607 Hamburg, Germany Vinorine synthase (VS, EC 2.3.1.160) is an acetyl transferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the Indian medicinal plant Rauvolfia serpentina. The enzyme catalyzes the reversible acetyl-CoA dependent formation of the ajmalan-type alkaloid vinorine from the sarpagine-type alkaloid 16-epi-vellosimine (Fig. 1) [1]. VS belongs to the BAHD (benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase) enzyme superfamily of acetyl transferases, which members are involved in biosynthesis of several important drugs (morphine, taxol, vincaleucoblastine and vincristine) and other products of secondary metabolism in plants. The synthase consists of 412 amino acids and has a molecular weight of 46.8 kDa. All conserved residues typical for the BAHD family are found in VS. Prominent features of this family are the HxxxD motif in the active site and the DFGWG motif near the C-terminus. The typical low overall sequence identity (25-34%) to other BAHD members might indicate a divergent evolution of the family from one ancestral gene [2]. O H C H OAc 16 AcCoA CoA N N N H H VS N H AcCoA CoA 16-epi- Vinorin Figure 1: Reaction catalyzed by vinorine synthase (VS) Vinorine synthase from R. serpentina was expressed in E. coli M15 cells using the plasmid pQE-2. The purification of His-tagged VS was carried out on a Ni-NTA column as previously described [3]. The His-tag was removed by dipeptidyl aminopeptidase digestion and VS was collected from the flowthrough of Ni-NTA. After following anion exchange chromatography VS was dialysed against 20 mM Tris-HCl, pH 7.5, 10 mM MSH and 1 mM EDTA and brought to a final concentration of 2 µg/µl for optimal crystallization results. The precipitant buffer contained 2.0 M ammonium sulphate, 2% PEG 400 and 100 mM Tris-HCl (pH 8.0) using the hanging drop method at 32°C. Crystals appeared after 3-5 days. The 3D-structure of VS we have recently described [4, 5] to a resolution of 2.6 Å. VS represents the first solved protein structure of the BAHD superfamily of acetyl transferases. The enzyme contains 13 α helices and 14 β strands, which can be divided into two domains of approximate equal size. In comparison with other acetyl transferases the position of the ligand acetyl-CoA was predicted by modelling (Fig. 2). At the beamline X13 a dataset of a co-crystal of VS including CoA was measured. The crystal diffracted to 3.0 Å. The rough position of the ligand was visible in the Fo-Fc electron density but not every atom could be recognised clearly. The Fo-Fc density appeared at different from the expected position. This unexpected result has to be proven by more experiments including dataset collection at better resolution. 335 modeled acetyl - CoA Fig. 2: Model of the structure of VS with the predicted position of its ligand acetyl-CoA. Dotted lines represent disordered regions. References [1] I. Gerasimenko, X. Y. Ma, Y. Sheludko, R. Mentele, F. Lottspeich, J. Stoeckigt, Bioorg. Med. Chem. 12, 2781 (2004) [2] B. St-Pierre, V. De Luca, Recent Advances in Phytochemistry, Evolution of Metabolic Pathways 34, 285 (2000) [3] A. Bayer, X. Y. Ma, J. Stöckigt, Bioorg. Med. Chem. 12, 2787 (2004) [4] X. Y. Ma, J. Koepke, S. Panjikar, G. Fritsch, J. Stoeckigt, J. Biol. Chem., 280, 13576 (2005) [5] X. Y. Ma, J. Koepke, A. Bayer, G. Fritzsch, H. Michel, J. Stoeckigt, Acta Crystallogr. D Biol Crystallogr. 61, 694 (2005) 336.
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