Identification and functional characterization of monofunctional ent-copalyl diphosphate and ent-kaurene synthases in white spruce reveal different patterns for diterpene synthase evolution for primary and secondary metabolism in gymnosperms

Abstract

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

Figure 1.

Comparison of the biosynthesis of gibberellins, as it is known in angiosperm and lower plants, with the biosynthesis of diterpene resin acids in conifers, a large group of gymnosperm trees. In conifers, the formation of diterpene resin acids involves bifunctional diTPS (e.g. abietadiene synthase) for the stepwise cyclization of GGPP into diterpenes such as abietadiene via a copalyl diphosphate intermediate that moves between the two active sites of the bifunctional diTPS (Peters et al., 2001). The products of the diTPS are subsequently oxidized by P450 to the resin acids. In contrast, gibberellin biosynthesis in angiosperms requires two monofunctional diTPSs to convert GGPP into ent-kaurene, which is subsequently modified by P450s. The two monofunctional diTPSs in angiosperm gibberellin biosynthesis are CPS and KS. In the lower plant P. patens, the CPS and KS activities are combined in a bifunctional diTPS similar to the bifunctional diTPS in conifer diterpene resin acid biosynthesis. Prior to this work, to our knowledge, it was not known if the formation of gibberellins in a gymnosperm involves two monofunctional diTPSs, as in angiosperms, or a bifunctional diTPS, as in gymnosperm diterpene resin acid biosynthesis and in P. patens gibberellin biosynthesis. (Figure adapted from Keeling and Bohlmann [2006a].)

Figure 2.

Structure of white spruce genomic DNA of BAC clones PGB08 and PGB09. The positions of the target genes PgCPS and PgKS in the pIndigoBAC-5 vector inserts are indicated. The scale bar represents 10 kb in the BAC sequences. Blue bars indicate the left and right arms of the pIndigoBAC-5 vector. In PGB08, the positions of gaps in the sequence assembly are indicated. Detailed gene structures are shown to scale below each BAC sequence, with green rectangles representing exons separated by introns.

Figure 3.

Amino acid alignment of monofunctional and bifunctional diTPSs of general and specialized metabolism, generated by MUSCLE. AtCPS, A. thaliana ent-CPS (NCBI accession no. AAA53632); PgCPS, P. glauca ent-CPS; PsCPS, P. sitchensis ent-CPS; PpCPS/KS, P. patens ent-CPS/ent-KS (BAF61135); AgAS, A. grandis abietadiene synthase (Q38710); AtKS, A. thaliana ent-KS (AAC39443); PgKS, P. glauca ent-KS; PsKS, P. sitchensis ent-KS. Amino acids with gray and black backgrounds indicate highly and completely conserved residues, respectively. Asp-rich motifs are indicated by underlines; a single underline indicates the DXDD motif necessary for protonation-initiated cyclization of GGPP to CPP, and a double underline indicates the DDXXD motif necessary for diphosphate ionization-initiated cyclization of CPP to the final diterpene products such as abietadiene and ent-kaurene.

Figure 4.

Unrooted phylogenetic tree of functionally characterized monofunctional and bifunctional diTPS proteins in general and specialized metabolism. AgAS, A. grandis abietadiene synthase (NCBI accession no. Q38710); AtCPS, A. thaliana ent-CPS (AAA53632); AtKS, A. thaliana ent-KS (AAC39443); CmCPS1, Cucurbita maxima ent-CPS1 (AAD04292); CmCPS2, C. maxima ent-CPS2 (AAD04293); CmKS, C. maxima ent-KS (AAB39482); GbLS, Ginkgo biloba levopimaradiene synthase (AAL09965); OsCPS1, O. sativa ent-CPS1 (BAD42449); OsCPS2, O. sativa ent-CPS2 (AAT11021); OsCPSsyn, O. sativa syn-CPS (AAS98158); OsKS1, O. sativa ent-KS (BAE72099); PaIso, P. abies isopimaradiene synthase (AAS47690); PaLAS, P. abies levopimaradiene/abietadiene synthase (AAS47691); PgCPS, P. glauca ent-CPS; PgKS, P. glauca ent-KS; PpCPS/KS, P. patens ent-CPS/ent-KS (BAF61135); PsCPS, P. sitchensis ent-CPS; PsKS, P. sitchensis ent-KS; PsaCPS, Pisum sativum ent-CPS (AAB58822); SrCPS, S. rebaudiana ent-CPS (AAB87091); SrKS1, S. rebaudiana ent-KS 1 (AAD34294); SrKS22, S. rebaudiana ent-KS 22 (AAD34295); TcTS, Taxus canadensis taxadiene synthase (AAR13860); ZmAn1, Z. mays ent-CPS1 (AAA73960); ZmAn2, Z. mays ent-CPS1 (AAT70083). The phylogenetic tree was prepared by protein alignment with MUSCLE, curation with Gblocks, phylogenetic analysis by PhyML (four rate substitution categories, gamma shape parameter optimized, Jones-Taylor-Thornton substitution model, and 100 bootstrap repetitions), and visualization with DrawTree. Asterisks indicate nodes supported by 80% or greater bootstrap values. The spruce CPS and KS proteins are positioned in the tree equidistant between the bifunctional diTPSs from gymnosperms and lower plants and the angiosperm monofunctional CPS and KS proteins, respectively. Without prior knowledge of the conserved DXDD and DDXXD motifs and functional characterization of these enzymes, it would not have been possible to predict whether the spruce enzymes were monofunctional or bifunctional.

Figure 5.

GC-MS analysis on a DB-WAX column of in vitro assays with purified recombinant proteins incubated with GGPP. TIC, Total ion current. To identify whether the white spruce PgCPS and PgKS enzymes were monofunctional or bifunctional enzymes, they were assayed with GGPP, alone or in combination with other enzymes. Neither PgCPS nor PgKS produced ent-kaurene when incubated alone with GGPP. However, when incubated with GGPP together or with complementary angiosperm monofunctional enzymes, ent-kaurene was produced, with identical mass spectral and elution characteristics to the product of bifunctional GfCPS/KS.

Figure 6.

Mass spectra of recombinant enzyme assay products. When incubated with GGPP, recombinant PgCPS+PgKS produced a product with identical elution (see Fig. 5) and mass spectral fragmentation patterns as the ent-kaurene produced by An2+OsKS1.

Figure 7.

Stereochemical analysis of enzyme assay products on a Cyclodex- β GC column by GC-MS. RIC, Reconstructed ion current of the molecular ion of kaurene. The assay product of PgCPS+PgKS incubated with GGPP eluted at the same retention time as an authentic standard of (–)-kaurene as well as the assay product of An2+OsKS1, which is known to produce (–)-kaurene when incubated with GGPP. When mixed with Wollemi pine extract, the assay product of PgCPS+PgKS did not coelute with the (+)-kaurene from Wollemi pine, confirming that PgCPS+PgKS produced (–)-kaurene when incubated with GGPP.

Figure 8.

Gene structures and schematic of proposed evolution of diTPS in plants. Roman numerals indicate intron numbers. Colored bars indicate exon sequences. Schematic, intron numbers, and exon coloring scheme are based upon Trapp and Croteau (2001b). Genomic DNA sequences compared are as follows: AgAS, A. grandis abietadiene synthase (NCBI accession no. AF326516); AtCPS, A. thaliana ent-CPS (AT4G02780); AtKS, A. thaliana ent-KS (AT1G79460.1); GbLS, G. biloba levopimaradiene synthase (AY574248); PgCPS, PgKS, PpCPS/KS, P. patens CPS/KS (scaffold_130:28184..34739, www.phytozome.org). The positions of the start codons in the white spruce genes PgCPS and PgKS are indicated by M. An ancestral plant bifunctional diTPS gene is postulated to have duplicated to give rise to the diTPSs of general and specialized metabolism. Neofunctionalization has given rise to the bifunctional diTPS of gymnosperm specialized metabolism (e.g. AgAS) while conserving the gene structure of the ancestral gene. In general metabolism, two paths have occurred. In the moss P. patens, a diTPS (PpCPS/KS) has remained bifunctional but has fewer conserved introns and one different intron from the ancestral gene. In the case of angiosperms and gymnosperms, further duplication and subfunctionalization of the ancestral gene have resulted in the monofunctional CPS and KS genes, although the gene structure has been well conserved except for the loss of one or two introns at the 5 ′ end of the gymnosperm genes.