Purification and Characterization of an Inducible Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspension Cultures

Abstract

An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a Km for famesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly-and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitortreated cells resulting from either the expression of multiple genes or multiple post-translational processing events.Sesquiterpenoids comprise a large and diverse class of isoprenoids found in plants, fungi, and select bacteria, and insects (6, 22). In plants, sesquiterpenoids are often associated with essential oils (22), and except for a limited number of cases, '

Gene family for an elicitor-induced sesquiterpene cyclase in tobacco.

“…GGA TAC ACT CAT CCG GAG AAA GTC TTA AAA CCT CAC ATT ATT AAC CTA CTT GTG GAC TCC The predicted translation product initiated at the first in-frame ATG following the transcription start site has a molecular mass of 56.8 kDa, consistent with the value of 60 kDa determined for the EAS protein by SDS/PAGE (11). Additional evidence that the gene encodes the previously purified EAS protein (11) was obtained by comparing the empirically determined N-terminal amino acid sequence of EAS with the predicted sequence as shown in Fig.…”

“…Tobacco EAS is a soluble, apparently cytoplasmic enzyme determined by SDS/PAGE to be a single polypeptide with a molecular mass of -60 kDa (11). The induction of EAS enzyme activity has been correlated with the absolute amount and the de novo synthesis of the enzyme protein (12).…”

Regulation of a Sesquiterpene Cyclase in Cellulase-Treated Tobacco Cell Suspension Cultures

“…Immunoblot analysis of proteins separated by SDS-PAGE was described previously (15,24,26). Specific immunoprecipitation of the radioactive cyclase protein produced in vivo or in vitro was similar to that of Mohnen et al (19).…”

Identification of the Uridine-Binding Domain of Sucrose-Phosphate Synthase (Expression of a Region of the Protein that Photoaffinity Labels with 5-Azidouridine Diphosphate-Glucose)

“…Polyclonal antibodies to purified leaf SPS or to a 26-kD portion of spinach SPS expressed in E. coli (see below) were prepared in BALB mice as described by Vogeli et al (1990). The immune serum produced against purified SPS was affinity purified by adsorption to and desorption from the 120-kD full-length or the 82-kD truncated SPS polypeptides (see below) bound to an Immobilon-P poly(viny1idenedifluoride) membrane (Millipore, Waltham, MA) as follows.…”