TY - JOUR
T1 - Protochlorophyllide oxidoreductase: An homology model examined by site-directed mutagenesis
AU - Townley, HE
AU - Sessions, RB
AU - Clarke, AR
AU - Dafforn, Timothy
AU - Griffiths, WT
PY - 2001/1/1
Y1 - 2001/1/1
N2 - An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme-binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the protein's fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations.
AB - An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme-binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the protein's fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations.
UR - http://www.scopus.com/inward/record.url?scp=0035882551&partnerID=8YFLogxK
U2 - 10.1002/prot.1098
DO - 10.1002/prot.1098
M3 - Article
C2 - 11455606
SN - 1097-0134
VL - 44
SP - 329
EP - 335
JO - Proteins: structure, function, and bioinformatics
JF - Proteins: structure, function, and bioinformatics
ER -