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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Authors: | Lianli Sun Martin Ruppert Yuri Sheludko Heribert Warzecha Yu Zhao Joachim Stöckigt |
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Affiliation: | College of Pharmaceutical Sciences, Department of Traditional Chinese Medicine and Natural Drug Research, Building of College of Pharmaceutical Sciences, Zijingang Campus, Zhejiang University, 310058 Hangzhou, PR China. |
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Abstract: | 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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences reported in this article have been submitted to the Gene Bank under Accession No: AY766462. |
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Keywords: | Aldo-keto reductase Functional expression Indole alkaloid metabolic network Perakine reductase Rauvolfia serpentina (Apocynaceae) |
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