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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.


Phylogenetic tree of putative kaurene synthase-(like) enzymes (KS(L)) and copalyl diphosphate synthases (CPS). The phylogenetic relationship of putative KS(L) and CPS synthases from P. trichocarpa to KS(L) and CPS from other plant species is shown. The tree was inferred with the neighbor-joining method and n = 1000 replicates for bootstrapping. Bootstrap values are shown next to each node. TPS-c and TPS-e, represent established TPS subfamilies [13]. PtTPS1 was used as an outgroup. KS: ent-kaur-16-ene synthase, SMS: stemar-13-ene synthase, LPPS: 8-hydroxy-copalyl diphosphate synthase, CPS: copalyl diphosphate synthase, Nt: Nicotiana tabacum, Cm: Cucurbita maxima, At: Arabidosis thaliana, Os: Oryza sativa, Pg: Picea glauca, Potri: Populus trichocarpa, Sm: Salvia miltiorrhiza, Pp: Physcomitrella patens
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Fig1: Phylogenetic tree of putative kaurene synthase-(like) enzymes (KS(L)) and copalyl diphosphate synthases (CPS). The phylogenetic relationship of putative KS(L) and CPS synthases from P. trichocarpa to KS(L) and CPS from other plant species is shown. The tree was inferred with the neighbor-joining method and n = 1000 replicates for bootstrapping. Bootstrap values are shown next to each node. TPS-c and TPS-e, represent established TPS subfamilies [13]. PtTPS1 was used as an outgroup. KS: ent-kaur-16-ene synthase, SMS: stemar-13-ene synthase, LPPS: 8-hydroxy-copalyl diphosphate synthase, CPS: copalyl diphosphate synthase, Nt: Nicotiana tabacum, Cm: Cucurbita maxima, At: Arabidosis thaliana, Os: Oryza sativa, Pg: Picea glauca, Potri: Populus trichocarpa, Sm: Salvia miltiorrhiza, Pp: Physcomitrella patens

Mentions: PtTPS17 and PtTPS18 share 89.4 % nucleotide similarity and are located on chromosome two and five, respectively, according to the available databases (www.phytozome.org). The high sequence similarity and the chromosomal locations of PtTPS17 and PtTPS18 indicate their origin through the recent genome duplication event described for poplar [22]. In a phylogenetic tree, the encoded proteins cluster together with characterized CPS proteins from other plants and are members of the TPS-c family (Fig. 1). Sequence motifs characteristic for class II TPS enzymes and important for CPS activity, such as the DxDD motif responsible for the initial protonation of the double bond and the EDxxD-like motif that coordinates the Mg2+ / diphosphate [13, 23], could be identified in both enzymes (Fig. 2). In addition, both proteins contained a conserved histidine residue that has been described to mediate sensitivity towards Mg2+ [24].Fig. 1


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)

Phylogenetic tree of putative kaurene synthase-(like) enzymes (KS(L)) and copalyl diphosphate synthases (CPS). The phylogenetic relationship of putative KS(L) and CPS synthases from P. trichocarpa to KS(L) and CPS from other plant species is shown. The tree was inferred with the neighbor-joining method and n = 1000 replicates for bootstrapping. Bootstrap values are shown next to each node. TPS-c and TPS-e, represent established TPS subfamilies [13]. PtTPS1 was used as an outgroup. KS: ent-kaur-16-ene synthase, SMS: stemar-13-ene synthase, LPPS: 8-hydroxy-copalyl diphosphate synthase, CPS: copalyl diphosphate synthase, Nt: Nicotiana tabacum, Cm: Cucurbita maxima, At: Arabidosis thaliana, Os: Oryza sativa, Pg: Picea glauca, Potri: Populus trichocarpa, Sm: Salvia miltiorrhiza, Pp: Physcomitrella patens
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Phylogenetic tree of putative kaurene synthase-(like) enzymes (KS(L)) and copalyl diphosphate synthases (CPS). The phylogenetic relationship of putative KS(L) and CPS synthases from P. trichocarpa to KS(L) and CPS from other plant species is shown. The tree was inferred with the neighbor-joining method and n = 1000 replicates for bootstrapping. Bootstrap values are shown next to each node. TPS-c and TPS-e, represent established TPS subfamilies [13]. PtTPS1 was used as an outgroup. KS: ent-kaur-16-ene synthase, SMS: stemar-13-ene synthase, LPPS: 8-hydroxy-copalyl diphosphate synthase, CPS: copalyl diphosphate synthase, Nt: Nicotiana tabacum, Cm: Cucurbita maxima, At: Arabidosis thaliana, Os: Oryza sativa, Pg: Picea glauca, Potri: Populus trichocarpa, Sm: Salvia miltiorrhiza, Pp: Physcomitrella patens
Mentions: PtTPS17 and PtTPS18 share 89.4 % nucleotide similarity and are located on chromosome two and five, respectively, according to the available databases (www.phytozome.org). The high sequence similarity and the chromosomal locations of PtTPS17 and PtTPS18 indicate their origin through the recent genome duplication event described for poplar [22]. In a phylogenetic tree, the encoded proteins cluster together with characterized CPS proteins from other plants and are members of the TPS-c family (Fig. 1). Sequence motifs characteristic for class II TPS enzymes and important for CPS activity, such as the DxDD motif responsible for the initial protonation of the double bond and the EDxxD-like motif that coordinates the Mg2+ / diphosphate [13, 23], could be identified in both enzymes (Fig. 2). In addition, both proteins contained a conserved histidine residue that has been described to mediate sensitivity towards Mg2+ [24].Fig. 1

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.