Isolation and characterization of two germacrene A synthase cDNA clones from chicory

Abstract

Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The K(m) value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.

Figure 1

Biosynthetic pathway of sesquiterpene lactones in chicory. Solid arrows indicate enzymatic steps previously demonstrated (de Kraker et al., 1998, 2001, 2002). 1, GAS; 2, germacrene A hydroxylase, 3, germacrene A alcohol dehydrogenase(s); 4, costunolide synthase; 5, further modifications. Broken arrows indicate postulated further steps (de Kraker et al., 2002).

Figure 2

Alignment of deduced amino acid sequences of chicory GASs, GASsh (=CiGASsh; GenBank accession no. AF498000) and GASlo (=CiGASlo; GenBank accession no. AF497999), with related plant sesquiterpene synthases: tomato germacrene B synthase (LeGBS; AAG41891), tomato germacrene C synthase (LeGCS; AAC39432), tomato germacrene D synthase (LeGDS; van der Hoeven et al., 2001), and tobacco 5-epi-aristolochene synthase (TEAS; T03714). The amino acid residues marked with an asterisk and three-letter code and position correspond to the position in TEAS and were hypothesized by Chappell and coworkers to be involved in catalysis of TEAS (Starks et al., 1997). Residues marked with # are also discussed in the text. The alignment was made using the ClustalX and Genedoc software.

Figure 3

Radio-GLC analysis of radiolabeled products formed from [³H]FDP in assays with protein extracts from transformed E. coli BL 21 (DE3) cells (Stratagene). A, Flame-ionization detector signal showing an unlabeled authentic standard of germacrene A. B and C, Radio traces showing enzymatic products of protein extracts from BL 21 (DE3) cells transformed with CiGASsh and CiGASlo, respectively. Insets show the mass spectra obtained using GC-MS analysis on an HP5-MS column of the same samples.

Figure 4

Western blot showing the expression of CiGASsh and CiGASlo in a number of chicory tissues. For each tissue and specific probe, 2 μg of total RNA was used (see “Materials and Methods” for more details).

Figure 5

Elution from MonoQ of two GAS proteins CiGASsh (○) and CiGASlo (▵), that were obtained using heterologous expression in E. coli and a partially purified (using Q-Sepharose anion-exchange chromatography) protein extract prepared from chicory (□). Enzymatic activity of eluting fractions was assayed using [³H]FDP as substrate and determining hexane soluble radiolabeled product formation using scintillation counting. Product identity was verified using radio-GLC.

Figure 6

Phylogenetic analysis of sesquiterpene synthases (from Van der Hoeven et al., 2001).

Figure 7

Molecular models of the two chicory GAS isoenzymes CiGASsh and CiGASlo. A, Detailed view of the active site residues of CiGASsh in (pale) green and CiGASlo in (pale) yellow and TEAS (T03714) in (pale) red. Pale colors indicate the amino acids with an identical position in the TEAS crystal structure and the GASs models. Bright colors indicate amino acids with differences in identity and/or spatial position that are discussed in the text. B, Detailed view of the active site residues of CiGASsh (green) and a selected number of amino acids (red) that have different physiochemical properties in the GASs compared with TEAS and that are discussed in the text. Molecular modeling was carried out using the Swiss-model service (http://www.expasy.ch/swissmod/; Peitsch, 1995, 1996; Guex and Peitsch, 1997) using the crystal structure of TEAS as a template. Models were rendered using POV-Ray for Windows (http://www.povray.org). Numbering follows the TEAS numbering (A) or the numbering of CiGASsh (B; also see Fig. 2).