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.

Photoprotective role of NADPH:protochlorophyllide oxidoreductase A

“…For example, upon treatment of synPOR with these agents, a decrease in the enzyme activity was observed (22). In the proposed structural models ( Fig.…”

“…The -amino group of the Lys side chain hereby is proposed to interact with the hydroxyl group of Tyr and to lower its pK a such that a proton can be donated during the catalytic mechanism. In addition, both amino acid residues are implicated in the formation of the POR photoactive state with Pchlide (22,(26)(27)(28).…”

“…The participation of one or two Cys residues in Pchlide binding has been proposed (22,29). SynPOR contains six Cys residues of which Cys-33, Cys-85, Cys-195, and Cys-222, henceforth referred to as Cys-1, Cys-2, Cys-3, and Cys-4, respectively, have counterparts in the PORA and PORB of barley ( Fig.…”

“…The GxxxGxG and YxxxK motifs implicated in NADPH binding and the conserved Cys residues (referred to as Cys-1, Cys-2, Cys-3, and Cys-4) are highlighted. Numbering refers to the full-length PORA and PORB polypeptides containing their NH 2-terminal transit peptides for plastid import (12) and synPOR, which does not contain a comparable NH 2-terminal extension (22). Identical amino acids are given in white letters on red background, whereas conserved residues are highlighted with red letters in black frames.…”

The First Catalytic Step of the Light-driven Enzyme Protochlorophyllide Oxidoreductase Proceeds via a Charge Transfer Complex

“…POR is a member of the "RED" superfamily of enzymes (reductases, epimerases, dehydrogenases) (2, 3), which catalyze NADP(H)-or NAD(H)-dependent reactions involving hydride and proton transfers (4). They have many structural features in common, and a three-dimensional homology model of the POR-Pchlide-NADPH ternary complex has been constructed using a member of this superfamily as a template (5).…”