Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH: protochlorophyllide oxidoreductase from Synechocystis.
NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic 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 Ni(2+)-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.
Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK. d.j.heyes@sheffield.ac.uk
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D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000201:enzymology*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine
D017931:DNA Primers; D011994:Recombinant Proteins; D014443:Tyrosine; D009249:NADP; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D008239:Lysine