Functional Analysis of Two Solanesyl Diphosphate Synthases fromArabidopsis thaliana

Abstract

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein.Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.Key words: isoprenoid; prenyltransferase; nonaprenyl diphosphate; plastoquinone; ubiquinone Plants have two major prenylquinones, plastoquinone and ubiquinone. 1) Although both share the structural feature of a trans-polyprenyl tail attached to the benzoquinone skeleton and have common oxidationreduction properties, their subcellular localization and biochemical roles are distinct. Plastoquinone exists in the thylakoid membrane of the chloroplast and acts as an electron carrier in the photosynthetic electron transfer reaction,2) whereas ubiquinone exists in the inner membrane of the mitochondrion and transfers electrons in the respiratory chain reaction.3)The hydrophobic tails of prenylquinones are C 30 -C 50 in length and serve as membrane anchors. The lengths of the polyprenyl side-chains differ between plastoquinone and ubiquinone, and even among plant species.4) It is thought that these polyprenyl chains are derived from C 30 -C 50 prenyl diphosphates formed by the consecutive condensation of isopentenyl diphosphate (IPP; C 5 ) with allylic diphosphate in the trans-configuration.5) As for the enzymes giving these precursors, several genes isolated from microorganisms have been well characterized, [6][7][8] but information about the plant-origin enzymes has been limited. Recently, we succeeded in the molecular cloning of Arabidopsis thaliana solanesyl diphosphate (SPP; C 45 ) synthase (At-SPS1; formerly designated At-SPS), which catalyzes the trans-type condensation of IPP to yield the C 45 product. 9) Enzymological analysis indicated that At-SPS1 utilizes both farnesyl diphosphate (FPP; C 15 ) and geranylgeranyl di...

Functional Modeling Identifies Paralogous Solanesyl-diphosphate Synthases That Assemble the Side Chain of Plastoquinone-9 in Plastids

“…The latter is not solely distinct from the AtSPS3 functional network but is also intimately linked to plastids. Although such a model fits with the demonstration that AtSPS2 is targeted to plastids, it conflicts with the previous assumption that AtSPS1 localizes to the endoplasmic reticulum (8,9).…”

“…12), and previous studies have indeed confirmed that AtSPS2 is targeted exclusively to chloroplasts (8,9,38,39). The localization of GFP-fused proteins demonstrates that this strict plastid localization holds true for AtSPS1 as long as the cognate gene is expressed under the control of its native promoter.…”

RNA-seq Analysis of Solanesol Biosynthesis Genes in <em>Nicotiana tabacum</em>

“…In the present study, both qRT-PCR and RNA-seq analyses indicated that the relative expression levels of NtSPS1 and NtSPS2 were significantly higher in leaves than in stems and roots (P < 0.05). To date, SPS homologs have been identified in A. thaliana [31][32][33], Hevea brasiliensis [34], Oryza sativa [35], and S. lycopersicum [36]. Hirooka et al [32] cloned two SPS genes from A. thaliana and found that the expression levels of AtSPS1 and AtSPS2 in leaves and stems were significantly higher than those in the roots, which is similar to the expression of NtSPS1 and NtSPS2 observed in the present study (Figure 3).…”

Farnesyl diphosphate synthase localizes to the cytoplasm of Trypanosoma cruzi and T. brucei

“…As pointed out in the introduction, the enzymes from the mevalonate pathway and particularly those of the isoprenoid biosynthetic route have been assigned to several different compartments in mammals and plants (20-23 Hugueney, 1996;Cunillera, 1997;Sanmiya, 1999;Biardi, 1996;Olivier, 2000;Hirooka, 2005;Leivar, 2005). Only a few enzymes of this pathway have been described in kinetoplastids (Montalvetti, 2001 andPena-Diaz, 2004;Ferella, 2006;Urbina, 2002;Ortiz-Gomez, 2006), and have been located to the glycosome, mitochondria, and cytoplasm (Ferella, 2006;Pena-Diaz, 2004 andOrtiz-Gomez, 2006, respectively).…”