Biosynthetic studies of naphthoquinones in impatiens balsamina root cultures

Abstract

Impatiens balsamina root cultures have been established in order to be used as a model for biosynthetic studies of lawsone-derived naphthoquinones. Chemical analysis of the ethyl acetate and methanolic extracts of the cultured roots by TLC revealed the presence of naphthoquinone and coumarin derivatives as major components. These compounds were isolated and their structures were elucidated by various spectroscopic data, as lawsone, 2-methoxy-1, 4-naphthoquinone, methylene-3, 3'-bilawsone (diphthicol), scopoletin, isofraxidin, 4, 4'-biisofraxidin and a plant sterol, spinasterol. Among these, methylene-3, 3'-bilawsone (bisnaphthoquinone) and 4, 4'-bilisofraxidin (biscoumarin) appeared to the new compounds. All these compounds couldb be effectively separted from one another by HPLC technique. Based on this tecnique, the chemical pattern and content of the secondary products produced by the cultured roots were investigated and compared with those of the intact plants (leaves and roots). The relationship between the root culture growth and the formation of each compound was also determined. Formation of the naphthoquinones was highly active in the late linear phase of the growth cycle. In vivo feeding experiments with [[superscript 14]C]alpha-ketoglutarate showed that the radiolabelled precursor was incorporated directly into lawsone and 2-methoxy-1, 4-naphthoquinone without detection of any radioactively labelled intermediates (e.g. o-succinylbenzoic acid). With cell-free extract prepared from the root cultures, [superscript 14]Calpha-ketoglutarate was also converted directly to 2-methoxy-1, 4-naphthoquinone, without detection of any labelled intermediates. These results suggest that various enzymes involved in the biosynthetic pathway of lawsone and 2-methoxy-1, 4-naphthoquinone in I. balsamina are organized as either multifunctional enzyme or multienzyme complex. The enzyme complex was eluted with void volume of superose 12 gel filtration column confirming its high molecular weight nature and complexity. The partially purified enzyme complex could use alpha-ketoglutarate and chorismic acid as substrates for the formation of 2-methoxy-1, 4-naphthoquinone, with coenzyme A, ATP and Mg[superscript 2+] as essentials for the enzyme activity. Lawsone-O-methyltransferase seemed not to be part of the enzyme complex. Methionine appeared to be the methyl source for 2-methoxy-1, 4-naphthoquinone, but not for the methylene bridge of methylene-3, 3'-bilawsone.