Properties of the recombinant beta subunit of glutamate synthase.
Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer. Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit. The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis. The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD. No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit. Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction. The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit). The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex. The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions. The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase [Vanoni, M.A., Edmondson, D.E., Zanetti, G. & Curti, B. (1992) Biochemistry 31, 4613-4623].
Vanoni
M A
MA
Dipartimento di Fisiologia e Biochimica Generali, Sezione di Chimica Biologica, Università degli Studi di Milano, Italy.
Verzotti
E
E
Zanetti
G
G
Curti
B
B
D000693:Anaerobiosis; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000032:analysis / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000378:metabolism; D005970:Glutamate Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013379:Substrate Specificity
D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D005970:Glutamate Synthase