Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH:: protochlorophyllide oxidoreductase from Synechocystis

NADPH: protochlorophyllide oxicloreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll blosynthetic pathway. Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes. A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the,Synechocystis POR enzyme. The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni2+-affinity column. The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity. The K-d value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced.