Structural studies of NADPH : protochlorophyllide oxidoreductase.

The light driven enzyme Protochlorophyllide Oxidoreductase (POR) is responsible for catalysing the reduction of the Cl7 – C18 double bond of the D ring of protochlorophyllide (Pchlide), in the presence of NADPH, forming chlorophyllide (Chlide). The reduction of
Pchlide involves a light-induced hydride transfer reaction from the pro-S face of nicotinamide adenine dinucleotide phosphate (NADPH) to the C'7 position, coupled to the addition of a proton to the C'8 position forming Chlide. The reaction catalysed by POR is a key step in chlorophyll biosynthesis and is essential in the development of chloroplasts. To achieve the maximum yield of Thermosynechococcus POR, E. coli were grown at 37 degC to an optical density of 1. 7 before being induced with 0.1 mM IPTG and grown for a further two hours at 25 degC. Purification of the over-expressed protein was done using a nickel affinity chromatography column followed by a cation exchange chromatography column. Purified protein could then be concentrated in an Amicon stirred cell concentrator to a maximum concentration of 0.3 mM. The NMR conditions were optimised, and experiments conducted at 45 degC with a sample in sodium phosphate buffer pH 5.5, containing 100 mM NaCl, 1 mM DTT and Roche complete protease provided the best quality HSQC spectrum, along with stabilising the protein for an indefinite period of time. Full deuteration of the protein did not yield the spectral improvements expected, and as a result the assignment spectra were acquired at 600 MHz using a l3C 15N labelled sample. A partial assignment of POR identified that, compared to the rest of the protein, the 33-residue loop insertion unique to POR is significantly more mobile and moves on a timescale similar to that associated with substrate binding and product release. Mutagenesis of the loop, whilst not affecting substrate binding, has profound effects on the enzyme activity, implicating the loop to be involved in some way with enzyme activity. Incubation of the enzyme with Pchlide and NADP+ results in the aggregation of the enzyme and the formation of a complex, which has been shown by EM and AUC to consist of approximately 10 units. This is analogous to the light harvesting Pchlide POR complex identified to exist in plants and proves that in vitro such complexes exist in other organisms other than monocotyledonous plants.