Characterisation of a Light Stimulated 'trans-cis' Isomerase involved in Coumarin Biosynthesis

Baban, Jamil (2001) Characterisation of a Light Stimulated 'trans-cis' Isomerase involved in Coumarin Biosynthesis. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The thesis is concerned with the possibility of a novel trans-cis 2,4-dihydroxycinnamic acid isomerase being involved in the biosynthesis of the benzopyrone nucleus of coumarins. The data are consistent with the presence of a light stimulated trans-cis isomerase located in the aerial parts of pea plants. The levels of umbelliferone in pea tissues were assessed by normal and reverse phase HPLC following extraction by lactone separation. The highest level of umbelliferone was found in aerial tissues (600 ng g FW^-1). Prior autoclaving of the tissues reduced this value to (54 ng g FW^-1) suggesting that most is present as the glycosylated precursor. Umbelliferone synthesis in the absence of light was demonstrated by comparison of the total levels of umbelliferone in pea seeds (46 ng g FW^-1) and etiolated seedlings (184 ng g FW^-1). The increase in umbelliferone cannot be accounted for by photoisomerisation and suggests an alternative mechanism of trans-cis isomerisation operates. The possibility of a trans-cis 2,4-dihyroxycinnamic acid isomerase was further investigated. An enzyme assay for trans-cis isomerase was developed. Substrate was prepared by a two-step procedure, which resulted in a yield equivalent to 11mg of pure 2,4-dihydroxycinnamic acid and its structure was confirmed by nuclear magnetic resonance and mass spectroscopy. Comparison of the absorption spectra of substrate (2,4-dihydroxycinnamic acid) and product (umbelliferone) revealed that a wavelength of 376 nm was suitable for the detection of umbelliferone formation. The pH optimum for trans-cis isomerase was pH 7.0. The activity of the trans-cis isomerase was inhibited by Cu⁺⁺, Ag⁺, Hg⁺⁺, Pb⁺⁺ ions and boiling, but stimulated by Mn⁺⁺, Co⁺⁺, Fe⁺⁺ ions, ATP and light. The molecular weight of the trans-cis was estimated to be in the range of 7-10 kDa by gel filtration. Overall, the data strongly suggest that umbelliferone biosynthesis in pea is facilitated by a low molecular weight, Mn⁺⁺ and light stimulated trans-cis isomerase.