NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation

Abstract

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus. This enzyme (His 6 -POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented. Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide. Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Plant Protochlorophyllide Oxidoreductases A and B

“…As shown in these studies by quantification of the acetoneextractable pigments, POR B as compared with POR A is more specific to PChlide by binding and photoconverting a higher amount of PChlide into Chlide (20). Moreover, the K m value of ϳ3 M obtained for POR A is of the same order as the values of 6.8 and 1.8 M reported for Synechocystis POR and the POR enzyme from Thermosynechococcus, respectively (35,36). On the other hand, there is a difference to the K m values of 0.27 and 0.47 M estimated for plant PORs from pea and oat (37,38).…”

Enzymology below 200 K: The kinetics and thermodynamics of the photochemistry catalyzed by protochlorophyllide oxidoreductase

“…However, the inclusion of either of the cofactors NADPH or NADP ϩ results in the detection of enzyme-bound Pchlide species with characteristic red-shifted fluorescence maxima, arising from the formation of a ternary complex. To ensure that all of the enzyme was in a nucleotide-bound form, the concentrations of NADPH (200 M) and NADP ϩ (500 M) used in these experiments were set much higher than the K d reported for cofactor binding (21). In the presence of NADP ϩ , a shoulder appears at the red edge of the main Pchlide band.…”

“…His-tagged POR from Synechocystis sp. PCC6803 was overproduced in Escherichia coli and purified, as described (21). The enzyme was purified further on a second column (2.5 cm ϫ 10 cm) containing red Sepharose CL-6B (Amersham Pharmacia) equilibrated with 50 mM Tris⅐HCl, pH 7.5͞1 mM DTT͞20 mM NaCl.…”

“…The recent use of heterologously expressed POR protein has provided an excellent opportunity to study the reaction in greater detail (7,(18)(19)(20)(21). As a result, an intermediate at 682 nm has been observed by using low-temperature fluorescence measurements (7,20), although careful inspection of the spectra reveals that only low levels of conversion (Ͻ10%) could be measured.…”

“…As a result, an intermediate at 682 nm has been observed by using low-temperature fluorescence measurements (7,20), although careful inspection of the spectra reveals that only low levels of conversion (Ͻ10%) could be measured. In the present work His-tagged POR from the cyanobacterium Synechocystis (21) has been analyzed by lowtemperature fluorescence and absorbance measurements. High levels of photoconversion (Ͼ95% of bound Pchlide) were obtained by using this enzyme, providing the opportunity to produce difference spectra with a very high signal-to-noise ratio.…”