Structural and Functional Studies of the Light-Dependent Protochlorophyllide Oxidoreductase Enzyme

The light dependent enzyme protochlorophyllide oxidoreductase (POR) is a key enzyme in the chlorophyll biosynthesis pathway, catalysing the reduction of the C17 C18 bond in protochlorophyllide (Pchlide) to form chlorophyllide (Chlide). This reaction involves the light-induced transfer of a hydride from the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor, followed by proton transfer from a catalytic tyrosine residue. Much work has been done to elucidate the catalytic mechanism of POR, however little is known about the protein structure. POR isoforms in plants are also notable as components of the prolamellar bodies (PLBs), large paracrystalline structures that are precursors to the thylakoid membranes in mature chloroplasts.
Bioinformatics studies have identified a number of proteins, related to POR, which contain similar structural features, leading to the production of a structural model for POR. A unique loop region of POR was shown by EPR to be mobile, with point mutations within this region causing a reduction in enzymatic activity. Production of a 2H, 13C, 15N labelled sample of POR for NMR studies has enabled significant advancement in the understanding of the protein structure. This includes the calculation of backbone torsion angles for the majority of the protein, in addition to the identification of multiple dynamic regions of the protein.
The protocol for purification of Pchlide, the substrate for POR, has been significantly improved, providing high quality pigment for study of the POR ternary complex. Various biophysical techniques have been used to study the macromolecular structure of these complexes, indicating the formation of large aggregates of the cyanobacterial enzyme induced by substrate binding, similar to PLBs. This has also led to the identification of ring structures, composed of 5 and 6 monomers of POR, which are likely to be the primary components of the cyanobacterial POR structures.