PLANT PHYSIOLOGY , Vol 101, Issue 3 1029-1038, Copyright © 1993 by American Society of Plant Biologists

METABOLISM AND ENZYMOLOGY

Intermolecular Nitrogen Transfer in the Enzymic Conversion of Glutamate to [delta]-Aminolevulinic Acid by Extracts of Chlorella vulgaris

S. M. Mayer, E. Gawlita, Y. J. Avissar, V. E. Anderson and S. I. Beale
Division of Biology and Medicine (S.M.M., Y.J.A., S.I.B.), and Department of Chemistry (E.G., V.E.A.), Brown University, Providence, Rhode Island 02912

[delta]-Aminolevulinic acid (ALA), the universal biosynthetic precursor of tetrapyrrole pigments, is synthesized from glutamate in plants, algae, and many bacteria via a three-step process that begins with activation by ligation of glutamate to tRNAGlu, followed by reduction to glutamate-1-semialdehyde (GSA) and conversion of GSA to ALA. The GSA aminotransferase step requires no substrate other than GSA. A previous study examined whether the aminotransferase reaction proceeds via intramolecular or intermolecular N transfer and concluded that the reaction catalyzed by Chlamydomonas extracts occurs via intermolecular N transfer (Y.-H.L. Mau and W.-Y. Wang [1988] Plant Physiol 86: 793-797). However, in that study the possibility was not excluded that the result was a consequence of N exchange among product ALA molecules during the incubation, rather than intermolecular N transfer during the conversion of GSA to ALA. Therefore, this question was reexamined in another species and with additional controls. A gel-filtered extract of Chlorella vulgaris cells was incubated with ATP, Mg2+, NADPH, tRNA, and a mixture of L-glutamate molecules, one-half of which were labeled with 15N and the other half with 13C at C-1. The ALA product was purified, derivatized, and analyzed by gas chromatography-mass spectrometry. A significant fraction of the ALA molecules was heavy by two mass units, indicating incorporation of both 15N and 13C. These results show that the N and C atoms of each ALA molecule were derived from different glutamate molecules. Control experiments indicated that the results could not be attributed to exchange of N atoms between glutamate or ALA molecules during the incubation. These results confirm the earlier conclusion that GSA is converted to ALA via intermolecular N transfer and extend the results to another species. The labeling results, combined with the results of kinetic and inhibitor studies, support a model for the GSA aminotransferase reaction in which a single molecule of GSA is converted to ALA via an enzyme-bound 4,5-diaminovaleric acid intermediate.