Cloning and Characterization of the Arabidopsis Thaliana SQS1 Gene Encoding Squalene Synthase

Abstract

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A fulllength Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQSl and SQS2) which are organized in a tandem array. SQSl and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQSl gene product, SQSl. RNA blot analysis has shown that the 1.6-kb SQSl mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQSl isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 11 1 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using ['Hlfarnesyl-P, and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQSl or chimeric Arubidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.Keywords: isoprenoid biosynthesis; Arabidopsis thaliana; yeast; squalene synthase; metabolic channeling.Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (farnesyl-P,) to produce squalene, the first committed precursor for sterol biosynthesis [I]. In plants, as in other eukaryotes, farnesyl-P, lies at a multiple branch point in the isoprenoid biosynthetic pathway [2]. Farnesyl-P, serves as a substrate for the first committed reactions of several branched pathways leading to the synthesis of isoprenoid compounds that are required for plant growth and development, such as phytosterols (membrane structure and function), dolichols (glycoprotein synthesis), ubiquinones (electron transport) or sesquiterpenoid phytoalexins (defense against pathogen attack). Farnesyl-P, also acts as a prenyl donor Enzymes. Squalene synthase, famesyl-diphosphate farnesyl transferase (EC 2.5.1.21); squalene epoxidase (EC 1.14.99.7); lanosterol synthase (EC 5.4.99.7).Note. The Arabidopsis SQS cDNA sequence reported here has been deposited in the GenBank and DDBJ databases under accession number X86692.in protein prenylation, a process that promotes membrane interactions and biological activities of a variety of cell proteins involved in signal transduction, membrane biogenesis and cell growth control [3, 41. In addition to the proposed role of SQS in the control of phytosterol biosynthesis, changes in SQS activity could also affect the flux of isoprenoid compounds down the various branches of t...

A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin

“…In yeast, disruption of the C-4 demethylation complex of Erg25p/Erg26p/Erg27p/Erg28p in erg28 mutants reduces ergosterol levels compared with wildtype strains (Gachotte et al, 2001), demonstrating the existence of protein complexes in triterpenoid biosynthesis (Mo et al, 2002) and the importance of proteinprotein interactions for metabolic flux in triterpenoid pathways. However, protein-protein interactions among enzymes in the early steps of triterpenoid biosynthesis had not been determined before, in spite of existing indirect evidence such as the C-terminal domain of Arabidopsis squalene synthase being required for efficient channeling of squalene to SQE (Kribii et al, 1997). The results shown here may pave the way for future investigations on the importance of SQEs and their interactions with OSCs in the production of specialized metabolites.…”

Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

“…Transformed cells were selected by AbA (Aureobasidin A) and after 3 days of growth were subcultured on YPD medium (Kribii et al 1997). The dammarenediol-II (50 mg/l) dissolved with ethanol was added to the medium as a substrate.…”

Cloning and expression analysis of squalene synthase, a key enzyme involved in antifungal steroidal glycoalkaloids biosynthesis from Solanum nigrum

“…The SnSS cDNA contains an ORF encoding a protein of 411 amino acids. By aligning deduced sequences of SnSS with other squalene synthases from taxonomically diverse species, an SnSS amino acid sequence is 79.7, 95.8, 80.2, 79.7, 81.2, 74.6, 69.1% identical to those in Glycyrrihiza glabra (11), Capsicum annuum (16), Panax ginseng (10), Solanum tuberosum (15), Euphorbia tirucalli (20), Arabidopsis thaliana (12,14,21), Oryza sativa (13), respectively.…”

“…5.242 squalene synthase is a potential site for the regulation of SGA production. Squalene synthase genes have been characterized in Nicotiana tabacum (9), Panax ginseng (10), Glycyrrhiza glabra (11), and other plants (12)(13)(14)(15)(16)(17)(18).…”