title: Intermolecular nitrogen transfer in the enzymic conversion of glutamate to delta-aminolevulinic acid by extracts of Chlorella vulgaris.
abstract: 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 tRNA(Glu), 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.
authors.affiliation: Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
authors.forename: S M
authors.initials: SM
authors.lastname: Mayer
authors.forename: E
authors.initials: E
authors.lastname: Gawlita
authors.forename: Y J
authors.initials: YJ
authors.lastname: Avissar
authors.forename: V E
authors.initials: VE
authors.lastname: Anderson
authors.forename: S I
authors.initials: SI
authors.lastname: Beale
mesh_terms: D000586:Amination; D000622:Aminolevulinic Acid / Q000378:metabolism*; D002708:Chlorella / Q000201:enzymology / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism
chemical_list: D005971:Glutamates; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; C057794:glutamate-1-semialdehyde; D000622:Aminolevulinic Acid; D009584:Nitrogen