Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii |
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Authors: | Peter Jurtshuk Linda McManus |
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Affiliation: | Department of Biology, University of Houston, Houston, Texas 77004 U.S.A. |
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Abstract: | l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD+, NADP+, FAD, or FMN for activity. O2, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R3 fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a2, a1, b1, c4 and c5) are reduced by the addition of l-glutamate. |
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Keywords: | DCIP dichlorophenolindophenol HQNO TTFB 4,4,4-trifluoro-1-(2-thienyl)-1,3-butadione NBT PMS phenazine methosulfate |
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