Studies on protoporphyrin biosynthetic pathway in Saccharomyces cerevisiae ; characterization of the tetrapyrrole intermediates

Abstract

An acellular extract of the yeast, Saccharomyces cerevisiae, incubated with ALA, is able to synthesize protoporphyrin from this precursor. Several tetrapyrrole intermediates were extracted from the medium and purified by silica gel chromatography. The chromatographic behaviour and the spectral properties of the isolated seven free carboxylic porphyrins (and of the corresponding esters), show that each product has a different carboxyle number, varying from eight (uroporphyrin) to two (protoporphyrin). The identification of five of them (octa- to tetracarboxymethyl-porphyrinester) is confirmed by mass spectrometry. The effect of physical factors (temperature, pH, time) on the protoporphyrin biosynthesis system indicates that the enzymes catalysing the first steps of the pathway (ALA leads to Coproporphyrin) are more stable than those catalysing the last steps (Coproporphyrin leads to Protoporphyrin). Results obtained with some enzymatic inhibitors (EDTA, OP, pCMB) show the sensitivity of the ALA dehydratase to OP and to pCMB (confirming therefore its nature as a metallo- and sulfhydryl enzyme) and also of the overall porphyrin synthesis system to these three agents.