Glutamate synthase from Chlamydomonas reinhardtii: Interaction studies with its substrate ferredoxin and molecular cloning

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able to form f unctional covalent complexes with its substrate ferredoxin (Fd), either wil d-type ((WT)Fd) or recombinant form ((r)Fd). However, when Fd carboxyl grou ps were chemically modified ((md)Fd), no complexes were detected and its ab ility to serve as electron donor for glutamate synthase activity was also d ecreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and a negative core in the helix alpha 1 are critical for Fd interaction wi th this enzyme and its functionality as electron carrier for glutamate synt hase. As a previous step to elucidate the specific positive charged residue s involved in glutamate synthase interaction with Fd, we have isolated a cD NA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii. The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structur ally more similar to higher plant enzymes than to the corresponding prokary otic GOGAT. Two conserved domains were present in this protein fragment, th e FMN-binding domain and the cysteines involved in the iron-sulfur cluster binding.