The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of theα/β hydrolase super family

Dogru, Emine; Warzecha, Heribert; Seibel, F.; Haebel, Sophie; Lottspeich, Friedrich; Stöckigt, Joachim

doi:10.1046/j.1432-1327.2000.01136.x

Cited by 81 publications

(101 citation statements)

References 34 publications

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“…Site-directed mutagenesis of (His) 6 PNAE was achieved using the QuikChange TM in vitro Site-Directed Mutagenesis Kit, according to the manufacturer's recommendations. For generation of the mutant enzymes, the following oligonucleotides were used: C20A: 5¢-CTGGTACACGGCGGAT GTCTCGGAGCTTGGATCTGG-3¢.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…After centrifugation (10 000 g, 20 min) total PNAE activity was determined in the supernatant. (His) 6 PNAE wild-type protein and the muteins were purified on self packed Ni 2+ resin columns (0.7 cm internal diameter · 1 cm). For the washing steps, the above mentioned buffer containing 20 and 50 mM imidazole, each of 20 mL was used and elution was performed with 250 mM imidazole.…”

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

“…For the determination of the relative molecular mass, 100 lL of (His) 6 PNAE wild-type protein, obtained from E. coli M15 cells (0.68 mgAEmL )1 ) was loaded onto a superdex column and fractionated (each 0.5 mL) with a 50 mM Na phosphate buffer pH 7.0 containing NaCl (150 mM). For comparison, the same procedure was repeated with 100 lL of the crude extract preparation of the enzyme (2.0 mgAEmL )1 ) from Rauvolfia plant cell suspension culture.…”

Section: Molecular Mass Determinationmentioning

confidence: 99%

“…The effects of the following inhibitors, in the mentioned concentrations, were checked on the pure (His) 6 PNAE enzyme fractions that were dialysed against 50 mM sodium phosphate buffer before use (pH 7.0, for 20 h at 4°C): AEBSF (1.0 mM, 4.0 mM), phenylmethanesulfonyl fluoride (1.0 mM), TPCK (200 lM), TLCK (120 lM), E-64 (25 lM), HgCl 2 (200 lM), DEPC (0.8-1.2 mM). The enzyme fractions, except DEPC, were preincubated with inhibitor for 1 h at 30°C.…”

Section: Inhibition Studiesmentioning

confidence: 99%

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Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Mattern-Dogru¹,

Ma²,

Hartmann³

et al. 2002

European Journal of Biochemistry

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In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the a/b hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By sitedirected mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the a/b hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the a/b hydrolase enzyme superfamily.Keywords: polyneuridine aldehyde esterase; active-site residues; site-directed mutagenesis; modelling; a/b hydrolase enzyme superfamily.Polyneuridine aldehyde esterase (PNAE, EC 3.1.1) is a central enzyme in a 10-step biosynthetic pathway expressed in the medicinal plant Rauvolfia serpentina Benth. ex Kurz. The pathway delivers the antiarrhythmic monoterpenoid indole alkaloid ajmaline [1,2]. From the enzymatic point of view, this is presently one of the most detailed investigated examples of a pathway leading to a natural product. Reactions of this complex biosynthetic sequence are catalyzed by membrane-bound and soluble enzymes such as cytochrome P450-dependent hydroxylases, a synthase, several reductases, a methyltransferase and a set of hydrolases including a b-glucosidase, an acetylesterase and the herein described methylesterase PNAE [3]. These enzymes exhibit a high degree of substrate specificity, PNAE being the most specific. PNAE is located in the middle of the biosynthetic chain starting from tryptamine and secologanin and catalyses the conversion of polyneuridine aldehyde into the next stable pathway intermediate, epivellosimine (Fig. 1). The enzyme has been detected in and partially characterized from cell suspension cultures of R. serpentina [4,5]. PNAE has also been highly enriched from cultured plant cells and the cDNA was recently functionally expressed in Escherichia coli [6]. Sequence alignment studies showed highest homologies to an esterase involved in pathogen defence in rice [7] that hydrolyses naphthol esters, and to hydroxynitrile lyases (HNLs) from Hevea brasiliensis [8] and Manihot esculenta [9]. The alignment suggested that PNAE is a new member of the a/b hydrolase superfamily that contains the putative catalytic triad serine, aspartic acid and histidine [10].For the present communication, a more detailed characterization of PNAE was performed by testing the influence of various inhibitors, HNL substrates and by structure elucid...

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Section: Site-directed Mutagenesismentioning

confidence: 99%

Section: Enzyme Expression and Purificationmentioning

confidence: 99%

Section: Molecular Mass Determinationmentioning

confidence: 99%

Section: Inhibition Studiesmentioning

confidence: 99%

See 2 more Smart Citations

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Mattern-Dogru¹,

Ma²,

Hartmann³

et al. 2002

European Journal of Biochemistry

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“…All biosynthetic transformations leading from strictosidine to ajmaline have been detected in enzyme fractions from Rauwolfia cell culture, though only six biosynthetic genes have been cloned and characterized (Figure 1). 4, 5, 6, 7, 8, 9, 10, 11 Several studies showed that vomilenine is an ajmaline biosynthetic intermediate that is produced through selective hydroxylation of vinorine at the C‐21 position by Rauwolfia cell culture extracts (Figure 1). 12, 13, 14 However, no enzyme isolated from R. serpentina has shown this activity 11…”

mentioning

confidence: 99%

Dual Catalytic Activity of a Cytochrome P450 Controls Bifurcation at a Metabolic Branch Point of Alkaloid Biosynthesis in Rauwolfia serpentina

et al. 2017

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Plants create tremendous chemical diversity from a single biosynthetic intermediate. In plant‐derived ajmalan alkaloid pathways, the biosynthetic intermediate vomilenine can be transformed into the anti‐arrhythmic compound ajmaline, or alternatively, can isomerize to form perakine, an alkaloid with a structurally distinct scaffold. Here we report the discovery and characterization of vinorine hydroxylase, a cytochrome P450 enzyme that hydroxylates vinorine to form vomilenine, which was found to exist as a mixture of rapidly interconverting epimers. Surprisingly, this cytochrome P450 also catalyzes the non‐oxidative isomerization of the ajmaline precursor vomilenine to perakine. This unusual dual catalytic activity of vinorine hydroxylase thereby provides a control mechanism for the bifurcation of these alkaloid pathway branches. This discovery highlights the unusual catalytic functionality that has evolved in plant pathways.

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Alkaloid Biosynthesis

O’Connor

2008

Wiley Encyclopedia of Chemical Biology

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How nature synthesizes complex secondary metabolites, or natural products, can be studied only by working within the disciplines of both chemistry and biology. Alkaloids are a complex group of natural products with diverse mechanisms of biosynthesis. This article highlights the biosynthesis of four major classes of plant‐derived alkaloids. Only plant alkaloids for which significant genetic information has been obtained were chosen for review. Isoquinoline alkaloid, terpenoid indole alkaloid, tropane alkaloid, and purine alkaloid biosynthesis are described here. The article is intended to provide an overview of the basic mechanism of biosynthesis for selected members of each pathway. Manipulation of these pathways by metabolic engineering is highlighted also.

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The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of theα/β hydrolase super family

Cited by 81 publications

References 34 publications

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Dual Catalytic Activity of a Cytochrome P450 Controls Bifurcation at a Metabolic Branch Point of Alkaloid Biosynthesis in Rauwolfia serpentina

Alkaloid Biosynthesis

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