The synthesis and properties of 4,5-dioxovaleric acid, a possible intermediate in the biosynthesis of 5-aminolaevulinic acid, and its in vivo formation in Scenedesmus obliquus.

The formation of 5-aminolaevulinic acid for chlorophyll biosynthesis in the pigment mutant C-2A' of the unicellular green alga Scenedesmus obliquus occurs via two pathways: the first and major pathway uses glycine and succinyl-CoA as precursors and the second, the C-5 pathway, uses the intact five C-atom skeleton of either glutamate or 2-oxoglutarate. The intermediates in this latter pathway of 5-aminolaevulinic acid biosynthesis are still a matter of controversy and discussion but, in this paper, we have demonstrated in this Scenedesmus mutant that 4,5-dioxovaleric acid occurs in the cells when illuminated in the presence of laevulinic acid and the amount of 4,5-dioxovaleric acid formed is dependent on the period of illumination. In a preliminary experiment we have shown, under similar conditions, that labelled 4,5-dioxovaleric acid can be obtained from both DL-[1-(14)C]glutamate and [2-(14)C]glycine. This suggests that 4,5-dioxovaleric acid is formed enzymically from the intact five C-atom skeleton of glutamate but may also arise from the deamination of ALA formed by the condensation of succinyl-CoA and glycine by the 5-aminolaevulinic acid synthase pathway. To obtain positive identification the naturally occurring, and also the labelled, 4,5-dioxovaleric acid were isolated as the more stable benzoquinoxaline derivative by condensation with 2,3-diaminonaphthalene and identified by comparison with the benzoquinoxaline derivative of an authentic sample of 4,5-dioxovaleric acid prepared by the hydrogenation of the ozonide of benzylidene laevulinic acid. The structures of the authentic sample of 4,5-dioxovaleric acid and its benzoquinoxaline derivative were confirmed by NMR and mass spectroscopy and both were further characterized by TLC and by infrared, ultraviolet and fluorescence spectroscopy.