The mechanism of the attachment of esterifying alcohol in bacteriochlorophyll a biosynthesis

The mechanism through which the C-17(3) carboxy group of bacteriochlorophyllide a is esterified to produce bacteriochlorophyll aphytyl of Rhodopseudomonas spheroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum was studied by using 5-aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms. The latter species was prepared by an exchange reaction in which 5-aminolaevulinate hydrochloride was heated in H218O in an autoclave. A method for the determination of the 18O content of the C-1 oxygen atoms of 5-aminolaevulinate was developed. As a prelude to the mechanistic work, a systematic study was undertaken to establish the optimal conditions under which a significant proportion of the bacteriochlorophyll a of the two photosynthetic organisms originated from the exogenously added 5-aminolaevulinate. It was found that, when Rps. spheroides and Rsp. rubrum were grown in the presence of about 0.15mM- and 1.2mM-5-aminolaevulinate respectively, 30-40% of their chlorophyll was derived from the added precursor. In these conditions, 5-amino[1,4-18O3]laevulinate was incorporated into bacteriochlorophyll aphytyl and bacteriochlorophyll ageranylgeranyl by the relevant organisms. The samples of chlorophylls were then hydrolysed with alkali to obtain phytol and geranylgeraniol, which were converted into the corresponding trimethylsilyl derivatives and analysed by gas chromatography-mass spectrometry. The data were used to deduce that the alcohols contained 90-95% of the 18O originally present at each of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results it is suggested that the ester bond at C-17(3) is formed, not by a chlorophyllase type of enzymic reaction, but by a process involving the nucleophilic attack by the C-17(3) carboxylate group of the chlorophyllide on the activated form of an isoprenyl alcohol.