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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.


Related in: MedlinePlus

GC-MS analysis of diterpenoids produced by recombinant PtTPS17, PtTPS18, PtTPS19 and PtTPS20. The enzymes were expressed in E. coli, extracted, partially purified, and incubated with 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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Fig3: GC-MS analysis of diterpenoids produced by recombinant PtTPS17, PtTPS18, PtTPS19 and PtTPS20. The enzymes were expressed in E. coli, extracted, partially purified, and incubated with 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: While no activity with GGPP could be observed for the putative KS(L) enzymes PtTPS19 and PtTPS20, neither alone nor in combinations with syn-CPS or n-CPS, diterpene product formation occurred when these enzymes were fed with GGPP in the presence of an ent-CPS. PtTPS19 converted ent-CPP into ent-kaurene and PtTPS20 converted this intermediate into 16α-hydroxy-ent-kaurane (86 %) and smaller amounts of ent-kaurene (8 %) and ent-isokaurene (6 %) (Fig. 3, Table 1). When PtTPS17 was incubated with GGPP, copalol was detected, as a result of the dephosphorylation of CPP. A comparison of the retention time of the copalol formed with those of authentic standards revealed that PtTPS17 produced either ent-CPP or normal-CPP (Additional file 1: Figure S2). However, the fact that PtTPS17 was able to support diterpene product formation when coupled with PtTPS19 or PtTPS20 confirmed that the enzyme mediated the formation of ent-CPP. Supplying PtTPS17 with different concentrations of Mg2+ did influence enzyme activity, with ent-CPP formation being higher at lower cofactor concentrations (Fig. 4). Despite the high sequence similarity to PtTPS17, no enzyme activity, neither with GGPP alone nor in combination with other CPS or KS, could be observed for PtTPS18 (Fig. 3). That a few amino acid mutations can affect enzyme activity has been shown for various terpene synthases (e.g. [26]). In all assays geranyllinalool formation could be detected, reflecting an unspecific dephosphorylation of the GGPP substrate. Attempts to verify enzyme activity in vivo by using crude protein extracts from poplar roots and leaves were not successful.Fig. 3


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)

GC-MS analysis of diterpenoids produced by recombinant PtTPS17, PtTPS18, PtTPS19 and PtTPS20. The enzymes were expressed in E. coli, extracted, partially purified, and incubated with 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4625925&req=5

Fig3: GC-MS analysis of diterpenoids produced by recombinant PtTPS17, PtTPS18, PtTPS19 and PtTPS20. The enzymes were expressed in E. coli, extracted, partially purified, and incubated with 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: While no activity with GGPP could be observed for the putative KS(L) enzymes PtTPS19 and PtTPS20, neither alone nor in combinations with syn-CPS or n-CPS, diterpene product formation occurred when these enzymes were fed with GGPP in the presence of an ent-CPS. PtTPS19 converted ent-CPP into ent-kaurene and PtTPS20 converted this intermediate into 16α-hydroxy-ent-kaurane (86 %) and smaller amounts of ent-kaurene (8 %) and ent-isokaurene (6 %) (Fig. 3, Table 1). When PtTPS17 was incubated with GGPP, copalol was detected, as a result of the dephosphorylation of CPP. A comparison of the retention time of the copalol formed with those of authentic standards revealed that PtTPS17 produced either ent-CPP or normal-CPP (Additional file 1: Figure S2). However, the fact that PtTPS17 was able to support diterpene product formation when coupled with PtTPS19 or PtTPS20 confirmed that the enzyme mediated the formation of ent-CPP. Supplying PtTPS17 with different concentrations of Mg2+ did influence enzyme activity, with ent-CPP formation being higher at lower cofactor concentrations (Fig. 4). Despite the high sequence similarity to PtTPS17, no enzyme activity, neither with GGPP alone nor in combination with other CPS or KS, could be observed for PtTPS18 (Fig. 3). That a few amino acid mutations can affect enzyme activity has been shown for various terpene synthases (e.g. [26]). In all assays geranyllinalool formation could be detected, reflecting an unspecific dephosphorylation of the GGPP substrate. Attempts to verify enzyme activity in vivo by using crude protein extracts from poplar roots and leaves were not successful.Fig. 3

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.


Related in: MedlinePlus