title: Protochlorophyllide oxidoreductase: a homology model examined by site-directed mutagenesis.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
authors.forename: H E
authors.initials: HE
authors.lastname: Townley
authors.forename: R B
authors.initials: RB
authors.lastname: Sessions
authors.forename: A R
authors.initials: AR
authors.lastname: Clarke
authors.forename: T R
authors.initials: TR
authors.lastname: Dafforn
authors.forename: W T
authors.initials: WT
authors.lastname: Griffiths
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D000458:Cyanobacteria / Q000201:enzymology*; D005453:Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017386:Sequence Homology, Amino Acid
chemical_list: D009249:NADP; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase