THE RECOMBINANT ALPHA-SUBUNIT OF GLUTAMATE SYNTHASE - SPECTROSCOPIC AND CATALYTIC PROPERTIES

As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzym e subunits separately in Escherichia coli. The beta 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 I of glu tamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This su bunit contains FMN as the flavin cofactor which exhibits the Propertie s 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 o f reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the s ite of reduction of the iminoglutarate formed on the addition of gluta mine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate s ynthase ct subunit contains tile [3Fe-4S] cluster of glutamate synthas e, as shown by low-temperature EPR spectroscopy experiments, The gluta mate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithi onite and methyl viologen) is present. The FMN moiety but not the [3Fe -4S] cluster of the subunit appears to participate in this reaction, F urthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holo enzyme. These findings support a model for glutamate synthase accordin g to which the enzymes prepared from various sources share a common gl utamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant e nzyme) but differ for the chosen electron donor. The pyridine nucleoti de-dependent forms of the enzyme have recruited a FAD-dependent oxidor eductase (the bacterial beta subunit) to mediate electron transfer fro m the NAD(P)H substrate to the glutamate synthase polypeptide. However , it appears that the presence of the enzyme beta subunit and/or of th e additional iron-sulfur clusters (Centers II and III) of the bacteria l glutamate synthase is required for communication between Center I (t he [3Fe-4S] center) and the FMN moiety within the alpha subunit, and f or ensuring coupling of glutamine hydrolysis to the transfer of the re leased ammonia molecule to 2-oxoglutarate in the holoenzyme.