Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Abstract

Plants and certain protists use cycloeucalenol cycloisomerase (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein.We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.Even distantly diverged organisms generally biosynthesize shared molecules by such similar routes that bacteria and yeast have been suitable systems for deducing much of human metabolism. Sterol biosynthesis provides an exception to this trend. Eukaryotes use two distinct pathways ( Fig. 1) to make structurally similar sterols (1). Animals and fungi cyclize oxidosqualene to the tetracyclic ⌬ 8 -triterpene lanosterol (2), which they metabolize further to tetracyclic membrane sterols (3)(4)(5). Plants cyclize instead to the pentacyclic cycloartenol (6, 7) and consequently must isomerize the cyclopropane ring to form ⌬ 8 tetracycles en route to tetracyclic sterols. Why plants use this two-enzyme route (8) rather than cyclizing directly to tetracyclic sterols using lanosterol synthase remains unknown.Although the enzyme has not been purified to homogeneity, cycloeucalenol cycloisomerase (also known as cycloeucalenolobtusifoliol isomerase) activity has been observed directly in both dicots (9) and monocots (10). In addition, the presence of an isomerase has been demonstrated indirectly in diverse plants (11)(12)(13)(14) and the protozoan Acanthamoeba polyphaga (15) by identifying cyclopropyl sterols after treatment with isomerase inhibitors. In D 2 O, the isomerase adds a deuterium to C-19 and abstracts hydrogen from C-8 (16, 17). Neither ATP nor NADPH enhances activity (8), and acidic ring opening seems the most likely mechanism. Because the nonenzymatic acid-catalyzed isomerization (18) requires severe conditions (10% H 2 SO 4 in refluxing isopropyl alcohol for 24 h), it is likely that the enzyme employs a metal or an unusually acidic amino acid residue to open the ring.As a prelude to investigating its catalytic mechanism and biological function, we have cloned and heterologously expressed the Arabidopsis thaliana cDNA that encodes cycloeucalenol cycloisomerase. EXPERIMENTAL PROCEDURESCloning the Cycloeucalenol Cycloisomerase cDNA-The A. thaliana CAS1 1 cDNA (19) cloned into the integrative yeast exp...

Biosynthesis of Phytosterol Esters: Identification of a Sterol O-Acyltransferase in Arabidopsis

“…8). Cycloartenol is the first tetracyclic sterol precursors in plant sterol biosynthesis pathway, and is considered an intermediate in the phytosterol biosynthesis pathway equivalent to lanosterol in yeast and animal systems (Benveniste 1986;Lovato et al, 2000;Kolesnikova et al, 2006;Suzuki et al, 2006). After cycloartenol, several major biochemical steps including methyltransferase, demethylation, and desaturation reactions need to take place before the final products of b-sitosterol and campesterol can be formed (Benveniste, 2004).…”

Biosynthesis of Cholesterol and Other Sterols

“…(233) The COI gene from Arabidopsis thaliana has been cloned and shown to encode a 36 kDa protein. (234) Notably, the COI cDNA compared with other plant sequences revealed that it was present in photosynthetic organisms. However, in no organism from prokaryotes or eukaryotes of a nonphotosynthetic lineage was significant identity to the plant gene evident.…”

TheSUD1Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity inArabidopsis

“…The selected mutants were cpi1-1, which causes the loss of function of the cyclopropylsterol isomerase gene (Lovato et al, 2000;Men et al, 2008), and fackel, which is mutated in a sterol C-14 reductase (Jang et al, 2000;Schrick et al, 2000). As shown in Supplemental Figure 4 online, no phenotypic recovery was observed when sud1-1 was introduced in the cpi1-1 and fk-x224 mutants.…”