S-Adenosyl-l-methionine:Loganic Acid Methyltransferase: A CARBOXYL-ALKYLATING ENZYME FROM VINCA ROSEA

Abstract

Abstract A partially purified enzyme from Vinca rosea was shown to catalyze the transfer of the methyl group of S-adenosyl-l-methionine to loganic acid forming loganin, an intermediate in the biosynthesis of indole alkaloids of this plant. Cell-free extracts, prepared in the presence of polyvinyl pyrrolidone, Na2S2O5, and dithiothreitol (DTT), were subjected to acetone precipitation, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. In assays with such preparations, DTT enhanced methyltransferase activity in contrast to mercaptoethanol, reduced glutathione, or cysteine. A combination of NADPH and FAD could partially replace DTT with the crude cell-free extract. Additional evidence for an active role of reduced sulfhydryl groups of the enzyme was obtained by inhibition studies with p-chloromercuribenzoate, iodoacetamide, and N-methylmaleimide. A divalent metal cation dependency was not detected. The apparent Km for loganic acid was 12.5 mm. Substrate specificity studies revealed that the rates of methylation of loganic and secologanic acids were comparable. Under the same assay conditions no significant methylation of 7-deoxyloganic, 7-epiloganic, or geniposidic acids was observed. Since the occurrence and biosynthesis of loganic and secologanic acids have also been demonstrated in V. rosea, both acids are thus implicated in the biosynthesis of the indole alkaloids.