Osoba, Olubukola Afolusho; (1994) Studies of the S-adenosyl-L-methionine methyltransferases from suspension cultures of ailanthus altissima (mill.) swingle (simaroubaceae). Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Ailanthus altissima cell suspension cultures produce significant levels of the biologically active canthin-6-one alkaloids as well as the coumarins scopoletin and isofraxidin . Detailed time course studies of two cell lines of A. altissima were carried out in order to determine the growth, alkaloid production patterns and l-hydroxycanthin-6- one methyltransferase (1-HMT) activities. These studies showed that cell line 1 produced l-methoxycanthin-6-one as the major constituent with canthin-6-one and 1- hydroxycanthin-6-one as minor constituents, whilst cell line 2 produced canthin-6-one as the major constituent with l-methoxycanthin-6-one and l-hydroxycanthin-6-one as minor constituents. The factors which could possibly regulate production of 1-methoxycanthin-6-one in cell cultures of A. altissima were discussed. 1-HMT was extracted from cell suspension cultures of A. altissima and purified to a single protein using standard techniques. The enzyme was characterised as a monomer of molecular weight 60,000 - 63,000 Dalton. The optimum conditions for enzyme activity were determined and optimum substrate concentrations were obtained from the Michealis-Menten constants for SAM and l-hydroxycanthin-6-one. Inhibitor studies revealed that 1-HMT was inhibited by the products of the methylation reaction, l-methoxycanthin-6-one and S-adenosylhomocysteine (SAH). 1-HMT was also inhibited by potassium cyanide and SH group inhibitors, iodoacetamide, N-ethylmaleimide and p-chloromercuric benzoate. The ability of the partially purified enzyme from various A. altissima cell lines to methylate 1 and 10-hydroxycanthin-6-one was also investigated. These studies indicate that A. altissima cell lines vary in their specificity to the two hydroxylated canthin-6-ones. The coumarin methyltransferase enzyme (CMT) from A. altissima cell suspension cultures was isolated and separated from the 1-HMT enzyme. Time course studies of the production of the identifiable methylated coumarins (scopoletin and isofraxidin ) as well as the corresponding methyltransferase activities were also carried out. A Preliminary investigation of the ability of CMT to methylate hydroxylated coumarins was also done. CMT was found to monomethyl ate aesculetin to yield scopoletin but not isoscopoletin. This enzyme also had the ability to further methylate isoscopoletin (not found in A. altissima cell cultures) to yield scoparone, but lacked the ability to further methylate scopoletin. CMT also monomethylated fraxetin to yield isofraxidin. Whilst this enzyme had the ability to further methylate fraxidin (not found in A. altissima cell cultures) to yield 6,7,8-trimethoxycoumarin, it did not further methylate isofraxidin . These results indicate that a pathway for the production of isofraxidin from aesculetin, via scopoletin and fraxetin, exists in A. altissima cell cultures.

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