Cloning of casbene synthase cDNA: evidence for conserved structural features among terpenoid cyclases in plants.

Abstract

A near-fll-length casbene synthase cDNA clone, pCS7, was isolated by using a partial cDNA clone, pCS4, to probe a g10 library constructed from poly(A)+ RNA from elicited castor bean dlings. The cDNA insert had a length of 1983 bases with a polyadenylate tail of 19 bases. Translation of the cDNA sequence revealed an open reading frame encoding a 601-aa protein with a predicted Mr of 68,960. Search of the GenBank data base with the deduced translation product revealed 42% identity and 65% similarity with 5-epiaristolochene synthe from tobacco and 31% identity and 53% simarity with limonene synthase from spearmint. Each of the three proteins catalyzes an intramolecular cyclization of a prenyl diphosphate substrate to a specific cyclic terpenoid hydrocarbon product. The proposed reaction mechanisms for the three catalytic processes share common chemical features, even though the products being formed are members of three different es of terpenoid compounds. Analysis of the alignment of the three proteins suggests that both primary and secondary structural elements are conserved. These smilarties suggest that the genes that encode terpenoid cyclization enzymes of this type in anglosperms have undergone divergent evolution from an ancestral progenitor gene. In support of this proposition, the locations offive ofthe six introns in the casbene synthase gene align very closely with those of the five introns in the 5-epi-aristolochene synthase gene.Casbene is a macrocyclic diterpene hydrocarbon that serves as a phytoalexin in castor bean (Ricinus communes L.) (1). Casbene synthase (CS; EC 4.6.1.7) (previously designated casbene synthetase) from castor bean seedlings catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to form the macrocyclic hydrocarbon in a single enzymatic step. The inducible enzyme activity is found in proplastids of germinating seedlings infected by the fungus Rhizopus stolonifer (2). The plant cells recognize oligogalacturonide fragments released from pectic components of the plant cell wall by a secreted fungal endopolygalacturonase as a signal to activate CS induction (3). CS activity reaches detectable levels after 5 hr of incubation with pectic fragments, with a peak occurring after 10-12 hr (4). The active enzyme consists of a single polypeptide and has an apparent Mr of 59,000 as determined by SDS/PAGE. Polyclonal antibodies to the purified enzyme have been used to isolate a partial cDNA clone from a Agtll library (5). RNA gel blots probed with this clone show that casbene synthase mRNA is undetectable in unelicited seedlings but accumulates to a maximum at 6 hr following elicitation. Nuclear run-on experiments indicate that control is exerted at the level of transcription.A full-length CS cDNA clone was isolated as part of an effort to characterize the gene structure in order to identify cis-acting elements involved in the induction of this gene by oligogalacturonide elicitors. This report describes the characterization of this clone § and the comparison of the primary struct...

Terpenoid metabolism.

“…It was shown to be part of a tobacco gene family, and two of the genes are arranged in tandem (Facchini and Chappell, 1992). The genomic structures of the exons of tobacco epi-aristolochene synthase and castor bean casbene synthase are remarkably similar (Mau and West, 1994).…”

“…It was shown to be part of a tobacco gene family, and two of the genes are arranged in tandem (Facchini and Chappell, 1992). The genomic structures of the exons of tobacco epi-aristolochene synthase and castor bean casbene synthase are remarkably similar (Mau and West, 1994).Comparing the deduced amino acid sequences of the terpene synthase genes reveals a fair degree of homology among them (Colbyet al, 1993;Mau and West, 1994;Chappell, 1995). These synthases, however, bear little or no homology with fungal or bacterial terpenoid cyclases or with any of the prenyltransferases.…”

“…Comparing the deduced amino acid sequences of the terpene synthase genes reveals a fair degree of homology among them (Colbyet al, 1993;Mau and West, 1994;Chappell, 1995). These synthases, however, bear little or no homology with fungal or bacterial terpenoid cyclases or with any of the prenyltransferases.…”

“…One other monoterpene synthase, a linalool synthase (Figure 2), has been cloned from C. breweri (E. Pichersky, personal communication). Three diterpene cyclases have also been cloned: casbene synthase from castor bean (Mau and West, 1994); abietadiene synthase ( Figure 2) from grand fir (B. Stofer Vogel and R. Croteau, unpublished results); and two genes for the A activity of ent-kaurene synthase (Figure 2), the Arabidopsis GA7 gene (Sun and Kamiya, 1994) and the maize An7 gene (Bensen et al, 1995). A sesquiterpene cyclase from tobacco, epi-aristolochene synthase (Figure 2), has also been cloned (Facchini and Chappell, 1992).…”

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

“…Besides Abies grandis, dozens of isoprenoid cyclase genes have been cloned from plants such as castor bean [31] Norway spruce [32,33], maize [34ϳ36], pine [37], ginkgo [38], snapdragon [39], grapevine [40], lotus [41], and tobacco [42]; some of these genes were isolated to investigate the relationship between isoprenoid production and their defensive effects against potential herbivores and pathogens at the molecular genetic level. Moreover, it was proved that among putative isoprenoid synthase genes discovered by whole genome sequence analysis and in silico analysis in Arabidopsis thaliana, at least six genes, namely, At3g25810, At1g61680, At4g16740, At2g24210, At3g25820, and At3g25830, encoded monoterpene synthases [43ϳ46].…”