Overexpression of higher plant nitrite reductase

The gene encoding the ferredoxin-dependent nitrite reductase of spinach chloroplasts (NiR; EC 1.7.7.1) has been isolated and sequenced (Back et ah, 1988). Although some information is available about the possible role of a few specific amino acids in the enzyme mechanism (Hirasawa et ah, 1994; Bellissimo et fl/.,1995; Dose et ah, 1997 and Dose, 1996), the absence of a 3-dimensional structure for the protein and the scarcity of data obtained with site-specific mutants have left considerable uncertainty about the enzyme mechanism. The work presented in this dissertation represents an attempt to address, in part, these issues by designing a system to over-express NiR in Escherichia coli and investigate the roles of the tryptophan residue at position 92 in the enzyme mechanism.