The Recombinant α Subunit of Glutamate Synthase:  Spectroscopic and Catalytic Properties

• Published in: Biochemistry (Easton) 1998-02, Vol.37 (7), p.1828-1838
• Main Authors: Vanoni, Maria A, Fischer, Federico, Ravasio, Sergio, Verzotti, Enrico, Edmondson, Dale E, Hagen, Wilfred R, Zanetti, Giuliana, Curti, Bruno
• Format: Article
• Language: English
• Subjects: Azospirillum brasilense - enzymology; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Biochemie; Biochemistry; Catalysis; Flavin Mononucleotide - chemistry; Flavin Mononucleotide - metabolism; Glutamate Synthase - biosynthesis; Glutamate Synthase - chemistry; Glutamate Synthase - genetics; Glutamate Synthase - isolation & purification; Glutamic Acid - biosynthesis; Glutaminase - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Spectrophotometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi972342w

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron−sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The β subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase α subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase:  reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (K d = 2.6 ± 0.22 mM), lack of reactivity with NADPH, reduction by l-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase α subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase α subunit catalyzes the synthesis of glutamate from l-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated α subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the α subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial β subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme β subunit and/or of the additional iron−sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the α subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammon