Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaloidal network of the plant Rauvolfia
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Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a “reverse-genetic” approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His6-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.
KeywordsAldo-keto reductase Functional expression Indole alkaloid metabolic network Perakine reductase Rauvolfia serpentina (Apocynaceae)
Polyacrylamide gel electrophoresis
Rapid amplification cDNA ends
Thin layer chromatography
We gratefully acknowledge Prof. Friedrich Lottspeich, Mrs. Isabella Mathes and Mr. Reinhard Mentele (Max-Planck-Institut für Biochemie, Martinsried, Germany) for amino acid sequence determination, the Deutsche Forschungsgemeinschaft (Bad-Godesberg, Germany) and Fonds der Chemischen Industrie (Frankfurt/Main, Germany) for financial support. Deutscher Akademischer Austauschdienst [Bonn, Germany, DAAD/D05/06969] and the China Scholarship Council [Beijing, People’s Republic of China CSC (2004) 3067] are acknowledged for support. We appreciate very much the help of Dr. Joachim Arend (Mainz, Germany) in measuring EI-mass spectra. GC-MS analyses were performed by SpectroData, company, Biebelsheim, Germany. We thank Dipl. Ing. Wilfried Löbel for measurements. We also thank Prof. Nikolaus Amrhein (ETH Zürich, Switzerland) for helpful discussions.
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