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One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar.

Irmisch S, Müller AT, Schmidt L, Günther J, Gershenzon J, Köllner TG - BMC Plant Biol. (2015)

Bottom Line: While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane.Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20.A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745, Jena, Germany. sirmisch@ice.mpg.de.

ABSTRACT

Background: Labdane-related diterpenoids form the largest group among the diterpenes. They fulfill important functions in primary metabolism as essential plant growth hormones and are known to function in secondary metabolism as, for example, phytoalexins. The biosynthesis of labdane-related diterpenes is mediated by the action of class II and class I diterpene synthases. Although terpene synthases have been well investigated in poplar, little is known about diterpene formation in this woody perennial plant species.

Results: The recently sequenced genome of Populus trichocarpa possesses two putative copalyl diphosphate synthase genes (CPS, class II) and two putative kaurene synthase genes (KS, class I), which most likely arose through a genome duplication and a recent tandem gene duplication, respectively. We showed that the CPS-like gene PtTPS17 encodes an ent-copalyl diphosphate synthase (ent-CPS), while the protein encoded by the putative CPS gene PtTPS18 showed no enzymatic activity. The putative kaurene synthases PtTPS19 and PtTPS20 both accepted ent-copalyl diphosphate (ent-CPP) as substrate. However, despite their high sequence similarity, they produced different diterpene products. While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane. Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20. A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles. Gene expression analysis revealed that the diterpene synthase genes characterized showed organ-specific expression with the highest abundance of PtTPS17 and PtTPS20 transcripts in poplar roots.

Conclusions: The poplar diterpene synthases PtTPS17, PtTPS19, and PtTPS20 contribute to the production of ent-kaurene and 16α-hydroxy-ent-kaurane in poplar. While ent-kaurene most likely serves as the universal precursor for gibberellins, the function of 16α-hydroxy-ent-kaurane in poplar is not known yet. However, the high expression levels of PtTPS20 and PtTPS17 in poplar roots may indicate an important function of 16α-hydroxy-ent-kaurane in secondary metabolism in this plant organ.

No MeSH data available.


Substrate specificity of PtTPS19 and PtTPS20. a Model of PtTPS19 showing their three domain structure (yellow: γ-domain, brown: β-domain, green: α-domain). b Model of the aligned active sites of PtTPS19 and PtTPS20. The conserved DDxxD motif is shown as blue sticks and the NDxxTxxxE/DDxxSxxxE motif is represented by purple sticks. Met607 of PtTPS19 and Thr607 of PtTPS20, which influence product outcome, are depicted as red and yellow sticks, respectively. Product formation of wild type enzymes (c) and enzymes possessing one amino acid exchange (d). The enzymes were expressed in E. coli, extracted, partially purified, and incubated with PtTPS17 and the substrate GGPP. Products were extracted with hexane and analyzed by GC-MS. 1, geranyllinalool; 2, copalol; 3, ent-kaurene; 4, ent-isokaurene; 5, 16α-hydroxy-ent-kaurane
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Fig5: Substrate specificity of PtTPS19 and PtTPS20. a Model of PtTPS19 showing their three domain structure (yellow: γ-domain, brown: β-domain, green: α-domain). b Model of the aligned active sites of PtTPS19 and PtTPS20. The conserved DDxxD motif is shown as blue sticks and the NDxxTxxxE/DDxxSxxxE motif is represented by purple sticks. Met607 of PtTPS19 and Thr607 of PtTPS20, which influence product outcome, are depicted as red and yellow sticks, respectively. Product formation of wild type enzymes (c) and enzymes possessing one amino acid exchange (d). The enzymes were expressed in E. coli, extracted, partially purified, and incubated with PtTPS17 and the substrate GGPP. Products were extracted with hexane and analyzed by GC-MS. 1, geranyllinalool; 2, copalol; 3, ent-kaurene; 4, ent-isokaurene; 5, 16α-hydroxy-ent-kaurane

Mentions: Although the PtTPS19 and PtTPS20 amino acid sequences were highly similar (99.1 %), their enzyme product profiles differed significantly. While PtTPS19 produced exclusively the diterpene hydrocarbon ent-kaurene, PtTPS20 mainly formed the diterpene-alcohol 16α-hydroxy-ent-kaurane (Fig. 3). To identify amino acids responsible for product specificity, homology-based structure models of PtTPS19 and PtTPS20 were constructed. Both models showed the three-domain structure (β, γ, and α domain) characteristic for the majority of plant DiTPS, with the catalytic site forming a deep pocket in the α domain (Fig. 5a,b; [23]). Only one amino acid differed in the active site of PtTPS19 compared to PtTPS20 (Fig. 2). While a methionine residue was present at position 607 in PtTPS19, the smaller, more polar threonine was situated at this position in PtTPS20 (Fig. 5b). Exchanging threonine 607 of PtTPS20 for methionine changed the product output of PtTPS20 completely. Instead of quenching the beyeran-16-yl cation by adding a water molecule and thus producing 16α-hydroxy-ent-kaurane, as observed for the wild type PtTPS20, the mutant enzyme catalyzed a deprotonation of the ent-kauranyl cation resulting in ent-kaurene formation comparable to PtTPS19 (Fig. 5d). Vice versa, the exchange of methionine 607 into a threonine in PtTPS19 resulted in a mutant able to produce mainly 16α-hydroxy-ent-kaurane and smaller amounts of ent-kaurene and ent-isokaurene in similar ratios as described for PtTPS20 (Fig. 5c, Table 1). The mutant PtTPS19 M607A produced also mainly 16α-hydroxy-ent-kaurane. However, exchanging the respective threonine 607 for alanine in PtTPS20 did not alter product specificity in comparison to the wild type enzyme (Table 1).Fig. 5


One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar.

Irmisch S, Müller AT, Schmidt L, Günther J, Gershenzon J, Köllner TG - BMC Plant Biol. (2015)

Substrate specificity of PtTPS19 and PtTPS20. a Model of PtTPS19 showing their three domain structure (yellow: γ-domain, brown: β-domain, green: α-domain). b Model of the aligned active sites of PtTPS19 and PtTPS20. The conserved DDxxD motif is shown as blue sticks and the NDxxTxxxE/DDxxSxxxE motif is represented by purple sticks. Met607 of PtTPS19 and Thr607 of PtTPS20, which influence product outcome, are depicted as red and yellow sticks, respectively. Product formation of wild type enzymes (c) and enzymes possessing one amino acid exchange (d). The enzymes were expressed in E. coli, extracted, partially purified, and incubated with PtTPS17 and the substrate GGPP. Products were extracted with hexane and analyzed by GC-MS. 1, geranyllinalool; 2, copalol; 3, ent-kaurene; 4, ent-isokaurene; 5, 16α-hydroxy-ent-kaurane
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Fig5: Substrate specificity of PtTPS19 and PtTPS20. a Model of PtTPS19 showing their three domain structure (yellow: γ-domain, brown: β-domain, green: α-domain). b Model of the aligned active sites of PtTPS19 and PtTPS20. The conserved DDxxD motif is shown as blue sticks and the NDxxTxxxE/DDxxSxxxE motif is represented by purple sticks. Met607 of PtTPS19 and Thr607 of PtTPS20, which influence product outcome, are depicted as red and yellow sticks, respectively. Product formation of wild type enzymes (c) and enzymes possessing one amino acid exchange (d). The enzymes were expressed in E. coli, extracted, partially purified, and incubated with PtTPS17 and the substrate GGPP. Products were extracted with hexane and analyzed by GC-MS. 1, geranyllinalool; 2, copalol; 3, ent-kaurene; 4, ent-isokaurene; 5, 16α-hydroxy-ent-kaurane
Mentions: Although the PtTPS19 and PtTPS20 amino acid sequences were highly similar (99.1 %), their enzyme product profiles differed significantly. While PtTPS19 produced exclusively the diterpene hydrocarbon ent-kaurene, PtTPS20 mainly formed the diterpene-alcohol 16α-hydroxy-ent-kaurane (Fig. 3). To identify amino acids responsible for product specificity, homology-based structure models of PtTPS19 and PtTPS20 were constructed. Both models showed the three-domain structure (β, γ, and α domain) characteristic for the majority of plant DiTPS, with the catalytic site forming a deep pocket in the α domain (Fig. 5a,b; [23]). Only one amino acid differed in the active site of PtTPS19 compared to PtTPS20 (Fig. 2). While a methionine residue was present at position 607 in PtTPS19, the smaller, more polar threonine was situated at this position in PtTPS20 (Fig. 5b). Exchanging threonine 607 of PtTPS20 for methionine changed the product output of PtTPS20 completely. Instead of quenching the beyeran-16-yl cation by adding a water molecule and thus producing 16α-hydroxy-ent-kaurane, as observed for the wild type PtTPS20, the mutant enzyme catalyzed a deprotonation of the ent-kauranyl cation resulting in ent-kaurene formation comparable to PtTPS19 (Fig. 5d). Vice versa, the exchange of methionine 607 into a threonine in PtTPS19 resulted in a mutant able to produce mainly 16α-hydroxy-ent-kaurane and smaller amounts of ent-kaurene and ent-isokaurene in similar ratios as described for PtTPS20 (Fig. 5c, Table 1). The mutant PtTPS19 M607A produced also mainly 16α-hydroxy-ent-kaurane. However, exchanging the respective threonine 607 for alanine in PtTPS20 did not alter product specificity in comparison to the wild type enzyme (Table 1).Fig. 5

Bottom Line: While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane.Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20.A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745, Jena, Germany. sirmisch@ice.mpg.de.

ABSTRACT

Background: Labdane-related diterpenoids form the largest group among the diterpenes. They fulfill important functions in primary metabolism as essential plant growth hormones and are known to function in secondary metabolism as, for example, phytoalexins. The biosynthesis of labdane-related diterpenes is mediated by the action of class II and class I diterpene synthases. Although terpene synthases have been well investigated in poplar, little is known about diterpene formation in this woody perennial plant species.

Results: The recently sequenced genome of Populus trichocarpa possesses two putative copalyl diphosphate synthase genes (CPS, class II) and two putative kaurene synthase genes (KS, class I), which most likely arose through a genome duplication and a recent tandem gene duplication, respectively. We showed that the CPS-like gene PtTPS17 encodes an ent-copalyl diphosphate synthase (ent-CPS), while the protein encoded by the putative CPS gene PtTPS18 showed no enzymatic activity. The putative kaurene synthases PtTPS19 and PtTPS20 both accepted ent-copalyl diphosphate (ent-CPP) as substrate. However, despite their high sequence similarity, they produced different diterpene products. While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane. Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20. A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles. Gene expression analysis revealed that the diterpene synthase genes characterized showed organ-specific expression with the highest abundance of PtTPS17 and PtTPS20 transcripts in poplar roots.

Conclusions: The poplar diterpene synthases PtTPS17, PtTPS19, and PtTPS20 contribute to the production of ent-kaurene and 16α-hydroxy-ent-kaurane in poplar. While ent-kaurene most likely serves as the universal precursor for gibberellins, the function of 16α-hydroxy-ent-kaurane in poplar is not known yet. However, the high expression levels of PtTPS20 and PtTPS17 in poplar roots may indicate an important function of 16α-hydroxy-ent-kaurane in secondary metabolism in this plant organ.

No MeSH data available.