Chem.-BioL Interactions Elsevier Publishing Company, Amsterdam Printed in The Netherlands

313

Possible involvement of cy tochrome P-450 in alkaloidal biosynthesis

BEVERLEY WILSON and STEN ORRENIUS Department of Biochemistry, University of Stockholm, Stockholm (Sweden}

Interest in the bioconversions and biogenesis of alkaloids, and of the alkaloids from Claviceps, in particular, has led us to a study of the involvement of cytochrome P-450 in the conversion of agroclavine, one of the principal clavine alkaloids. Metabolic studies have shown that microsomes from rat liver were capable of converting agroclavine to noragroclavine, the major conversion product, and elymoclavine, the next step in the biosynthetic sequence. The difference spectrum resulting from the introduction of agrodavine into a microsomal suspension is a modified Type II. Because of a general lack of association between Type II spectra and metabolism, the spectrum was studied further. It was shown that in the presence of hexobarbital at low levels, a modified Type II spectrum of much greater amplitude resulted but was composed of 2 overlapping peaks. In the presence of aniline a complex spectrum emerged which could be seen to be the result of a Type I and a Type II spectrum. With imidazole as the modifier, a similarly complex difference spectrum was obtained.

On the basis of these results, it was concluded that the modified Type II spectrum of agroclavine was the result of 2 spectra, a Type I and a Type II spectrum. Probing deeper into the question of the origin of secondary cellular metabolites, we then investigated the interaction of tryptophan, a precursor of the clavine alkaloids and ergot alkaloids in general, with cytochrome P-450 from microsomes of phenobarbital-treated rat liver. Again, a modified Type II spectrum emerged at rather high levels of L-tryptophan. With gradual increases in tryptophan concentration, this spectrum reached a point at which it suddenly increased markedly in amplitude and became more symmetrical, and equally quickly, a further increase in substrate level resulted in a complex spectrum which previous experience with agroclavine suggested to be a result of both a Type I and a Type II spectrum. Addition of the same modifiers as were used with agroclavine showed that all modifiers altered the spectra of lower levels of L-tryptophan to more closely resemble the spectrum of the modifier, but that regardless of the modifier, a concentration of tryptophan could be attained at which the marked spectral amplification could be achieved. Type II modifiers markedly delayed the establishment of such a spectrum, whereas hexobarbital did not. Regardless of the modifier, however, the spectrum obtained following the "clearing" concentration always had a similar shape and could be shown to contain both Type I and Type II components. The interaction of high levels of tryptophan with cytochrome P-450, as evidenced by the difference spectra, makes a study of the possible involvement of cytochrome P-450 in the metabolism of tryptophan of interest. Such studies are currently under way.

Aided by the Swedish Cancer Society and the Swedish Medical Research Council.

Chem.-BioL Interactions, 3 (1971) 313