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

Abstract

BackgroundLabdane-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.ResultsThe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0647-6) contains supplementary material, which is available to authorized users.

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

“…Here, we have shown that a single-point mutation that causes an amino acid substitution close to the active site dramatically alters product specificity in both SAD1 and LUP1, so uncovering hidden functional diversity in these triterpene synthases. It is conceivable that nature explores alternate modes of cyclization through such single mutational steps, as has been suggested for diterpene synthases (36)(37)(38)(39)(40)(41)(42)(43). A dedicated cyclase that makes epDM as its major product has not been reported before our work to our knowledge.…”

Biosynthetic Origin of the Ether Ring in Platensimycin

“…42 A similar product specificity switch was seen in Pt TPS20 from Populus trichocarpa when a T607M (analogous to A710 in Pp CPS/KS) mutation altered the typically (16 R )- ent -kauran-16-ol dominant (~86%) product profile to 100% ent -kaurene. 43 The monofunctional Bj KS from the Proteobacterium Bradyrhizobium japonicum solely produces ent -kaurene (Figure S7), and its crystal structure revealed that two bulky and hydrophobic residues, Y136 and L140 (analogous to A710 in Pp CPS/KS), likely block water from accessing the cyclization intermediate. 44 PtmT3 is the first (16 R )- ent -kauran-16-ol synthase from bacteria, providing an excellent opportunity to study diterpene cyclization and product determination from a bacterial DTS.…”

Separate Pathways Contribute to the Herbivore-Induced Formation of 2-Phenylethanol in Poplar

“…It has been shown that single amino acid changes can dramatically alter the specificity or activity of enzymes involved in specialized metabolism (e.g. Johnson et al, 2001;Köllner et al, 2004;Junker et al, 2013;Irmisch et al, 2015;Fan et al, 2017), suggesting that novel enzyme functions may arise in relatively short time periods by the accumulation of only a few nucleotide mutations. PtAADC1 and PtAAS1 provide an example of rapid evolutionary changes.…”