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Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

Krithika R, Srivastava PL, Rani B, Kolet SP, Chopade M, Soniya M, Thulasiram HV - Sci Rep (2015)

Bottom Line: Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value.One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial.These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Chemical Biology Unit, Division of Organic Chemistry, CSIR- National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008.

ABSTRACT
Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

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Total ion chromatograms (TICs) for 10-hydroxygeraniol dehydrogenase (Cr10HGO) catalyzed reactions with (a) 10-hydroxygeraniol and NADP+, (b) 10-oxogeraniol and NADP+, (c) 10-oxogeraniol and NADPH, (d)10-oxogeranial and NADPH, (e) 10-hydroxygeranial and NADP+, (f) 10-hydroxygeranial and NADPH as substrates. The peaks represent (2) 10-hydroxygeraniol, (3) 10-oxogeraniol, (4) 10-hydroxygeranial, and (5) 10-oxogeranial.
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f2: Total ion chromatograms (TICs) for 10-hydroxygeraniol dehydrogenase (Cr10HGO) catalyzed reactions with (a) 10-hydroxygeraniol and NADP+, (b) 10-oxogeraniol and NADP+, (c) 10-oxogeraniol and NADPH, (d)10-oxogeranial and NADPH, (e) 10-hydroxygeranial and NADP+, (f) 10-hydroxygeranial and NADPH as substrates. The peaks represent (2) 10-hydroxygeraniol, (3) 10-oxogeraniol, (4) 10-hydroxygeranial, and (5) 10-oxogeranial.

Mentions: Gas chromatography and mass spectrometric analyses of the reaction products after incubation of purified Cr10HGO protein with 10-hydroxygeraniol in the presence of NADP+ resulted in the formation of 10-oxogeranial along with 10-oxogeraniol and 10-hydroxygeranial as minor products (Fig. 2a and Supplementary Fig. S3). The formation of these products was further confirmed by comparing the fragmentation pattern as well as co-injection studies using corresponding synthesized compounds, 10-oxogeranial, 10-oxogeraniol and 10-hydroxygeranial. Further, Cr10HGO efficiently converted 10-oxogeraniol and 10-hydroxygeranial into 10-oxogeranial in the presence of NADP+. However, when NADPH was used as cofactor, 10-hydroxygeraniol was found to be the major enzymatic product with substrates, 10-oxogeraniol, 10-hydroxygeranial and 10-oxogeranial (Fig. 2) indicating that the Cr10HGO mediated reaction (Fig. 3) is reversible. The NADP+ dependent oxidoreductase protein purified from R. serpentina catalyzes dehydrogenation of nerol and geraniol in an efficient manner compared to10-hydroxygeraniol14. Similarly, the oxidoreductase purified from Nepeta racemosa18 also showed better activity towards geraniol, nerol and 10-hydroxynerol than towards 10-hydroxygeraniol. The recently reported 8-HGO, which encodes the NADP+ dependent oxidoreductase from C. roseus carries out the dehydrogenation of 10-hydroxygeraniol and also other acyclic monoterpenes19, but does not possess much sequence similarity with Cr10HGO. In contrast to these observations, monohydroxy terpene derivatives such as geraniol, nerol, and farnesol were found to be poor substrates for Cr10HGO as compared to the reported 8HGO19 (Supplementary Table S2 and Supplementary Fig. S4). Studies on the effects of temperature on Cr10HGO mediated reaction revealed that the Cr10HGO activity was found to be optimum at 30°C. The apparent Km values were found to be 1.50 μM for 10-hydroxygeraniol, 1.0 μM for 10-oxogeraniol and 10-hydroxygeranial at saturated concentrations of NADP+ (Supplementary Figures S5–S11, Supplementary Table S3). The kinetic studies also indicated that among NAD+/(H) and NADP+/(H), the latter was found to be the preferred coenzyme for Cr10HGO (Table 1).


Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

Krithika R, Srivastava PL, Rani B, Kolet SP, Chopade M, Soniya M, Thulasiram HV - Sci Rep (2015)

Total ion chromatograms (TICs) for 10-hydroxygeraniol dehydrogenase (Cr10HGO) catalyzed reactions with (a) 10-hydroxygeraniol and NADP+, (b) 10-oxogeraniol and NADP+, (c) 10-oxogeraniol and NADPH, (d)10-oxogeranial and NADPH, (e) 10-hydroxygeranial and NADP+, (f) 10-hydroxygeranial and NADPH as substrates. The peaks represent (2) 10-hydroxygeraniol, (3) 10-oxogeraniol, (4) 10-hydroxygeranial, and (5) 10-oxogeranial.
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f2: Total ion chromatograms (TICs) for 10-hydroxygeraniol dehydrogenase (Cr10HGO) catalyzed reactions with (a) 10-hydroxygeraniol and NADP+, (b) 10-oxogeraniol and NADP+, (c) 10-oxogeraniol and NADPH, (d)10-oxogeranial and NADPH, (e) 10-hydroxygeranial and NADP+, (f) 10-hydroxygeranial and NADPH as substrates. The peaks represent (2) 10-hydroxygeraniol, (3) 10-oxogeraniol, (4) 10-hydroxygeranial, and (5) 10-oxogeranial.
Mentions: Gas chromatography and mass spectrometric analyses of the reaction products after incubation of purified Cr10HGO protein with 10-hydroxygeraniol in the presence of NADP+ resulted in the formation of 10-oxogeranial along with 10-oxogeraniol and 10-hydroxygeranial as minor products (Fig. 2a and Supplementary Fig. S3). The formation of these products was further confirmed by comparing the fragmentation pattern as well as co-injection studies using corresponding synthesized compounds, 10-oxogeranial, 10-oxogeraniol and 10-hydroxygeranial. Further, Cr10HGO efficiently converted 10-oxogeraniol and 10-hydroxygeranial into 10-oxogeranial in the presence of NADP+. However, when NADPH was used as cofactor, 10-hydroxygeraniol was found to be the major enzymatic product with substrates, 10-oxogeraniol, 10-hydroxygeranial and 10-oxogeranial (Fig. 2) indicating that the Cr10HGO mediated reaction (Fig. 3) is reversible. The NADP+ dependent oxidoreductase protein purified from R. serpentina catalyzes dehydrogenation of nerol and geraniol in an efficient manner compared to10-hydroxygeraniol14. Similarly, the oxidoreductase purified from Nepeta racemosa18 also showed better activity towards geraniol, nerol and 10-hydroxynerol than towards 10-hydroxygeraniol. The recently reported 8-HGO, which encodes the NADP+ dependent oxidoreductase from C. roseus carries out the dehydrogenation of 10-hydroxygeraniol and also other acyclic monoterpenes19, but does not possess much sequence similarity with Cr10HGO. In contrast to these observations, monohydroxy terpene derivatives such as geraniol, nerol, and farnesol were found to be poor substrates for Cr10HGO as compared to the reported 8HGO19 (Supplementary Table S2 and Supplementary Fig. S4). Studies on the effects of temperature on Cr10HGO mediated reaction revealed that the Cr10HGO activity was found to be optimum at 30°C. The apparent Km values were found to be 1.50 μM for 10-hydroxygeraniol, 1.0 μM for 10-oxogeraniol and 10-hydroxygeranial at saturated concentrations of NADP+ (Supplementary Figures S5–S11, Supplementary Table S3). The kinetic studies also indicated that among NAD+/(H) and NADP+/(H), the latter was found to be the preferred coenzyme for Cr10HGO (Table 1).

Bottom Line: Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value.One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial.These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Chemical Biology Unit, Division of Organic Chemistry, CSIR- National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008.

ABSTRACT
Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

Show MeSH
Related in: MedlinePlus