cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus

• Published in: Biochimica et biophysica acta Vol. 1626; no. 1; pp. 97 - 101
• Main Authors: Akamine, Satomi, Nakamori, Kazuki, Chechetka, Svetlana A., Banba, Mari, Umehara, Yosuke, Kouchi, Hiroshi, Izui, Katsura, Hata, Shingo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2003
• Subjects: Aspartic Acid - analysis; Base Sequence; Binding Sites; Cloning, Molecular; DNA Mutational Analysis; DNA, Complementary; Farnesyl-Diphosphate Farnesyltransferase - biosynthesis; Farnesyl-Diphosphate Farnesyltransferase - chemistry; Farnesyl-Diphosphate Farnesyltransferase - genetics; Farnesyl-Diphosphate Farnesyltransferase - metabolism; In Situ Hybridization; Lotus - anatomy & histology; Lotus - enzymology; Lotus - genetics; Lotus japonicus; Molecular Sequence Data; Mutagenesis, Site-Directed; Phylogeny; Plant Roots - anatomy & histology; Plant Roots - enzymology; Plant Roots - genetics; Recombinant protein; RNA, Messenger - biosynthesis; RNA, Plant - biosynthesis; Site-directed mutagenesis; Recombinant protein; In situ hybridization; Lotus japonicus; Site-directed mutagenesis
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(03)00042-3

A full-length cDNA for squalene synthase was isolated from Lotus japonicus, a model leguminous plant. The transcript was abundant in roots, symbiotic root nodules, and shoots, in that order. In situ hybridization revealed that the mRNA level is high in expanding root cells but low in dividing root tip ones. The transcript is also abundant in vascular bundles and the basal portions of mature nodules. L. japonicus squalene synthase has an unusual Asp residue near the active site, where mammalian enzymes have Gln, and replacement of the Gln by Glu has been reported to cause severe inactivation. Site-directed mutagenesis of the L. japonicus enzyme and assaying in vitro showed that this Asp residue can be substituted by not only Gln but also Glu, suggesting that the local structure of plant squalene synthases is different from that of mammalian enzymes.