Information on EC 1.3.1.99 - iridoid synthase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.3.1.99
-
RECOMMENDED NAME
GeneOntology No.
iridoid synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(6E)-8-oxogeranial + NAD(P)H + H+ = cis-trans-nepetalactol + NAD(P)+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
Michael cyclization
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
secologanin and strictosidine biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
8-oxogeranial:NAD(P)+ oxidoreductase (cyclizing, cis-trans-nepetalactol forming)
Isolated from the plant Catharanthus roseus. The reaction may involve cyclization via a Diels-Alder or Michael reaction. Iridoids are involved in the biosynthesis of many indole alkaloids. The cyclic hemiacetal is readily hydrolysed to the corresponding dial.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
plant material is harvested from field plants of cv. Leccino from the Olive Cultivar Collection of CNR-Institute of Biosciences and Bio-resources, Perugia, Italy
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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truncation of the N-terminus has minimal impact on catalytic efficiency
metabolism
physiological function
additional information
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the oxyanion hole anchors geranic acid directly above the NADP+ cofactor, an orientation in which the pro-S hydride of NADPH is poised for addition to C3. Such a hydride transfer generates the S-stereocenter at this carbon, which is observed in the final product, further indicating a catalytically relevant orientation of the inhibitor
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(1E,3S,6E)-9,9-difluoro-3,7-dimethylnona-1,6,8-trien-1-ol + NADPH + H+
(1E,3S,6E)-8-chloro-3,7-dimethylocta-1,6-dien-1-ol + NADP+
show the reaction diagram
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6 is an artificial substrate analogue
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-
?
(2E,6E)-9,9-difluoro-3,7-dimethylnona-2,6,8-trienal + NADPH + H+
(2E,6E)-8-chloro-3,7-dimethylocta-2,6-dienal + NADP+
show the reaction diagram
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5 is an artificial substrate analogue
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-
?
(6E)-10-oxogeranial + NADH + H+
cis-trans-nepetalactol + NAD+
show the reaction diagram
(6E)-10-oxogeranial + NADPH + H+
cis-trans-nepetalactol + NADP+
show the reaction diagram
-
-
-
?
(6E)-8-oxogeranial + NAD(P)H + H+
cis-trans-nepetalactol + NAD(P)+
show the reaction diagram
(6E)-8-oxogeranial + NADPH + H+
cis-trans-nepetalactol + NADP+
show the reaction diagram
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-
-
-
?
(R)-10-oxocitronellal + NAD(P)H + H+
?
show the reaction diagram
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about 10% activity compared to 10-oxogeranial
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ir
(S)-10-oxocitronellal + NAD(P)H + H+
?
show the reaction diagram
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less than 5% activity compared to 10-oxogeranial
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ir
10-hydroxygeranial + NADPH + H+
10-hydroxycitronellol + NADP+
show the reaction diagram
recombinant enzyme in transgenic Saccharomyces cerevisiae within a reconstructed nepetalactol synthesis pathway, overview
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?
10-oxogeranial + NADH + H+
cis-trans-nepetalactol + NAD+
show the reaction diagram
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100% activity
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ir
10-oxogeranial + NADPH + H+
cis-trans-nepetalactol + NADP+
show the reaction diagram
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100% activity
-
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ir
10-oxogeraniol + NAD(P)H + H+
?
show the reaction diagram
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less than 10% activity compared to 10-oxogeranial
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ir
6,7-dihydro-10-oxogeranial + NAD(P)H + H+
?
show the reaction diagram
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about 25% activity compared to 10-oxogeranial
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ir
citral + NAD(P)H + H+
?
show the reaction diagram
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about 90% activity compared to 10-oxogeranial
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ir
geranial + NADPH + H+
citronellol + NADP+
show the reaction diagram
recombinant enzyme in transgenic Saccharomyces cerevisiae within a reconstructed nepetalactol synthesis pathway, overview
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(6E)-10-oxogeranial + NADH + H+
cis-trans-nepetalactol + NAD+
show the reaction diagram
-
-
-
-
?
(6E)-10-oxogeranial + NADPH + H+
cis-trans-nepetalactol + NADP+
show the reaction diagram
K7WDL7
-
-
-
?
(6E)-8-oxogeranial + NAD(P)H + H+
cis-trans-nepetalactol + NAD(P)+
show the reaction diagram
(6E)-8-oxogeranial + NADPH + H+
cis-trans-nepetalactol + NADP+
show the reaction diagram
-
-
-
-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
geranic acid
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resembles the proposed enolate intermediate and acts non-competitively, geranic acid binding pocket and binding structure, overview
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0819 - 0.485
(1E,3S,6E)-8-chloro-3,7-dimethylocta-1,6-dien-1-ol
0.0019 - 0.026
(6E)-8-oxogeranial
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.4 - 8.1
(1E,3S,6E)-8-chloro-3,7-dimethylocta-1,6-dien-1-ol
1.4 - 6.1
(6E)-8-oxogeranial
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
13 - 99
(1E,3S,6E)-8-chloro-3,7-dimethylocta-1,6-dien-1-ol
14
(6E)-8-oxogeranial
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pH 7.0, temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004
geranic acid
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pH 7.0, 25C, recombinant wild-type enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature
23 - 40
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assay at
25
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assay at
30
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified enzyme in apo form and with NADP+ bound, and as ternary complex with NADP+ and inhibitor geranic acid, sitting drop vapor diffusion, mixing of 300 nl of 7 mg/ml protein in 5 mM MOPS buffer, pH 7.0, 40 mM NaCl, 5 mM tris(2-carboxyethyl)phosphine, and 2 mM NADP+, with 300 nl of well solution, whose composition depends on the enzyme-ligand complex, overview, 20C, X-ray diffraction structure determination and analysis at 1.90 A, 1.40 A, and 1.75 A resolution, respectively. Crystallization requires truncation of the N-terminus, which has minimal impact on catalytic efficiency, molecular replacement using the structure of progesteron 5beta reductase from Digitalis lanata, PDB ID 2V6Gas search model
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purified iridoid synthase in apo state and in complex with NADP+ and 8-oxogeranial, hanging drop vapor diffusion method, mixing of 0.001 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 10 mM DTT, with 0.001 ml of reservoir solution containing 0.1 M HEPES, pH 7.5, 1.98 M ammonium sulfate, and 1% PEG 400, and equilibration against 1 ml of reservoir solution, to obtain the enzyme/cofactor/substrate ternary complex crystals, 15 mg/ml protein in a buffer of 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 10 mM DTT is incubated with 2 mM of NADP+ and 1 mM of 8-oxogeranial for 1 h before co-crystallization, 16C, one week, X-ray diffraction structure determination and analysis at 2.24 A and 2.71 A resolution, respectively, molecular replacement method using the DlP5betaR/NADP+ binary complex, PDB ID 2V6G, and the apo structure as search models, respectively
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purified recombinant wild-type and truncated mutant enzymes in apoforms and complexed with either NAD+, NADPH, or NAD+/10-oxogeranial, NAD+-enzyme crystals are soaked in a 10-oxogeranial solution, X-ray diffraction structure determination and analysis
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, tag cleavage by a protease, and anion exchange chromatography, followed by gel filtration
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recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, tag cleavage through 3C protease, followed by ultrafiltration, gel filtration, anion exchange chromatography, and ultrafiltration, to over 95% purity
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recombinant wild-type and truncated mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the complete cDNA including 5' and 3' UTRs, phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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DNA and amino acid sequence analysis, recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli
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expressed in Escherichia coli
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iridoid synthase gene, recombinant expression in transgenic Saccharomyces cerevisiae strain ERG20 with a chromosomal knock-in of the erg20K197E mutation, recombinant coexpression with diverse genes from Catharanthus roseus and Saccharomyces cerevisiae, overview
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recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli
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recombinant expression of wild-type and truncated mutant enzymes
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sequence comparisons and phylogenetic analysis, secoiridoid genes are co-expressed, i.e. in particular, iridoid synthase (OeISY) and 1,4-reductase 1 (Oe1,4-R1) grouped together with 8-hydroxygeraniol oxidoreductase (Oe8HGO), iridoid oxidase (OeIO), and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (OeDXR), geraniol 8-hydroxylase (OeG8H), and secologanin synthase-like (OeSLS-like3)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A346I
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site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
F149M
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site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
F342A
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site-directed mutagenesis, inactive mutant
K146S
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site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
S349F
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site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
Y178A
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site-directed mutagenesis, inactive mutant
YY178F
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site-directed mutagenesis, the mutant shows a 400fold decrease in kcat/Km and a 30fold decrease in kcat compared to the wild-type, but still yields cyclized product
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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potential of iridoid synthase to synthesize other cyclic compounds from nonnatural substrates
additional information
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the enzyme binds the 10-oxogeranial substrate in a transoid-O1-C3 conformation, the transoid 10-oxogeranial structure also serves as a model for beta-face hydride attack in progesterone 5beta-reductases and is of general interest in the context of asymmetric synthesis