Arabidopsis thaliana isoprenyl diphosphate synthases produce the C₂₅ intermediate geranylfarnesyl diphosphate

Abstract

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short-chain all-trans (E) prenyl diphosphates geranyl diphosphate (GDP, C10 ), farnesyl diphosphate (FDP, C15 ) or geranylgeranyl diphosphate (GGDP, C20 ). In the genome of Arabidopsis thaliana, 15 trans-product-forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC-MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C25 ) instead of GGDP as their major product in enzyme assays performed in vitro. Over-expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N-terminal to the first aspartate-rich motif is responsible for C25 versus C20 product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C20 ) as a product and a larger R group (Met) resulting in GFDP (C25 ).

A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum

“…Kinetic analysis using Lineweaver-Burk plots revealed that the recombinant GFDPS preferred allylic diphosphates as substrates in the order of GGDP = FDP > GDP > DMADP ( Table 2). The kinetic constants for GGDP and IDP were in the same range as those for other IDSs (Schmidt and Gershenzon, 2007;Nagel et al, 2015).…”

“…Lc-GFDPS shares 52 to 62% identity with Arabidopsis GFDPSs and GFDPSs from other Brassicaceae plants (Nagel et al, 2015;Wang et al, 2015), but only 33 and 34% identity with M. mazei GFDPS and A. pernix GFDPS, respectively. In Arabidopsis, GFDPS activity is associated with a change in the 5th amino acid in front of the first aspartate-rich motif from Met in GGDPS to Ala or Ser in GFDPS.…”

“…The GFDPS enzymes reported from Brassicaceae plants and archaea all produce GGDP or GDP as minor products, in addition to GFDP (Tachibana et al, 2000;Ogawa et al, 2010;Nagel et al, 2015;Wang et al, 2015). Lc-GFDPS preferred GGDP or FDP as its allylic cosubstrate and showed much less activity with GDP and DMADP, which is similar to the substrate specificity of Arabidopsis IDS9 and M. mazei GFDPS but distinct from that of Arabidopsis IDS7 and A. pernix GFDPS (Tachibana et al, 2000;Ogawa et al, 2010;Nagel et al, 2015;Wang et al, 2015). These results point to differences in the GFDPS of various organisms and are consistent with independent recruitment of this enzyme in different plant lineages.…”

“…For instance, mangicols, a class of sesterterpenoids with antiinflammatory activity isolated from the marine fungus Fusarium heterosporum, have been demonstrated to use GFDP as their biosynthetic precursor through feeding of labeled sodium acetate (Renner et al, 2000). However, little is known about the biosynthesis of plant sesterterpenoids, except for an analysis of the formation of the linear C 25 precursor GFDP in the Brassicaceae (Nagel et al, 2015;Wang et al, 2015).…”

“…Plant sesterterpenoids have been shown to exhibit a broad range of biological activities, serving as insect antifeedant (Luo et al, 2010(Luo et al, , 2011(Luo et al, , 2013a(Luo et al, , 2013bLi et al, 2013) and antifungal (Luo et al, 2010;Li et al, 2013) agents, as cytostatic agents against human lung cancer cell (Rowland et al, 2001), as inhibitors of tubulin tyrosine ligase (Dal Piaz et al, 2009, as prolylendopeptidase inhibitors (Choudhary et al, 2004a), and as enhancers of interleukin-2 gene expression (Kawahara et al, 1999). However, our knowledge of sesterterpenoid biosynthesis and evolution in plants is rather limited, and genes and enzymes involved in plant sesterterpenoid formation have only very recently been reported from species of the Brassicaceae (Nagel et al, 2015;Wang et al, 2015). It would be interesting to learn if these genes arose from genes forming larger, better known groups of terpenoids, such as those of C 20 diterpenoids, by duplication, divergence, and neofunctionalization (Pichersky and Lewinsohn, 2011;Kliebenstein and Osbourn, 2012), or if they were introduced from other sources, such as microorganisms, through horizontal gene transfer (Copley and Dhillon, 2002;Richards et al, 2006;Emiliani et al, 2009).…”