Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Xueyan Ma,Juergen Koepke,Santosh Panjikar,Günter Fritzsch,Joachim Stöckigt

doi:10.1074/jbc.m414508200

Abstract

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

Highlights

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia
Vinorine synthase belongs to the benzylalcohol acetyl, anthocyanin-O-hydroxy-cinnamoyl, anthranilate-N-hydroxy-cinnamoyl/benzoyl, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine
Ajmaline is an antiarrhythmic drug from the Indian plant Rauvolfia serpentina, which has been known as a medicinal plant for about 3000 years

Summary

EXPERIMENTAL PROCEDURES

Overexpression, Purification, and Crystallization of VS—VS was subcloned into the pQE-2 vector and overexpressed in E. coli. Data Collection and Processing—Both SeMet VS and native VS crystals were cryoprotected by addition of 20 –25% glycerol to the precipitant buffer before being flash-cooled in a stream of cold nitrogen at 100 K. Native data and multi-wavelength anomalous diffraction data from SeMet VS crystals measured at three different wavelengths around the selenium absorption edge were collected using synchrotron radiation on the BW7A beamline of the European Molecular Biology Laboratory at the DORIS storage ring of the Deutsches Elektronen-Synchrotron (Hamburg, Germany). The platform provided the correct selenium sites and an interpretable map with a partial ␣-helical model containing 167 of 842 residues. Phases were extended to 2.6 Å using data from the native crystal by density

SeMet inflection

RESULTS

DISCUSSION