NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, puri¢cation and preliminary characterisation

Abstract NADPH:protochlorophyllide oxidoreductase (POR)catalyses the light-dependent reduction of protochlorophyllide tochlorophyllide, a key regulatory reaction in the chlorophyllbiosynthetic pathway. POR from the cyanobacterium Synecho-cystis has been overproduced in Escherichia coli with ahexahistidine tag at the N-terminus. This enzyme (His 6 -POR)has been purified to homogeneity and a preliminary character-isation of its kinetic and substrate binding properties is presented.Chemical modification experiments have been used to demon-strate inhibition of POR activity by the thiol-specific reagentN-ethyl maleimide. Substrate protection experiments reveal thatthe modified Cys residues are involved in either substrate bindingor catalysis. s 2000 Federation of European Biochemical So-cieties. Published by Elsevier Science B.V. All rights reserved.Key words: NADPH:protochlorophyllide oxidoreductase;Chlorophyll biosynthesis; Light-dependent;Chemical modi¢cation; Synechocystis1. IntroductionIn angiosperms, the reduction of protochlorophyllide(Pchlide) to chlorophyllide (Chlide) is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) [1].Light is essential for the activity of POR and consequentlythis reaction is an important regulatory step in the chlorophyllbiosynthetic pathway. In addition to POR, non-£owering landplants, algae and cyanobacteria possess a light-independentPchlide reductase, consisting of three separate subunits [2].In these organisms Chlide synthesis can occur in the darkand it appears that the activity of the light-dependent enzymeis important for maximum chlorophyll synthesis [3^5].In higher plants, there are two POR isoforms, termedPORA and PORB, which are diierentially regulated. PORAaccumulates to high levels in the dark and is rapidly degradedupon illumination whereas PORB remains at a constant levelthroughout illumination [6]. In contrast, some organisms thatcan form chlorophyll in the dark, such as Chlamydomonasrheinhardtii [7] and Synechocystis sp. PCC6803 [8], containonly a single POR-encoding gene. Furthermore, PORs fromcyanobacteria do not accumulate when they are grown in thedark and are only ever present at very low levels [9], in bothplasma and thylakoid membrane fractions [10]. This is prob-ably due to the fact that these organisms possess the light-independent reductase in addition to POR, which prevents theaccumulation of Pchlide and the ternary complex. This lowlevel of enzyme together with the presence of interfering pig-ments has previously made PORs from cyanobacteria muchmore di⁄cult to study than the enzymes from higher plants.Cyanobacteria are generally good model organisms for thestudy of chlorophyll biosynthesis as they share many of thefeatures of higher plants whilst retaining the advantages ofbeing prokaryotes. In recent years, heterologous expressionof Synechocystis chlorophyll biosynthetic enzymes has beeninvaluable for the study of several steps in the pathway. Com-plementation of bacteriochlorophyll-minus Rhodobacter mu-tants led to the identi¢cation of Synechocystis chl genes encod-ing magnesium protoporphyrin IX methyl transferase [11], thelight-dependent protochlorophyllide oxidoreductase [12] andthe geranylgeranyl chlorophyll reductase [13]. Expression ofthe chlH, I and D genes in Escherichia coli led to the charac-terisation of the steady state kinetics of magnesium chelatasefrom Synechocystis [14^16]. Similarly, Oster et al. [17] success-fully expressed the chlG gene encoding chlorophyll synthetasein E. coli.The reaction catalysed by POR has been studied extensivelyand a number of spectroscopic forms of bound Pchlide havebeen identi¢ed [18]. However, to date, the catalytic mecha-nism of POR has not been elucidated. Comparisons of theamino acid sequence of POR with other sequences in thedatabase have indicated that it is a member of the short-chaindehydrogenase family of enzymes [19,20] and mutational anal-ysis of two residues conserved in all such dehydrogenasesdemonstrated that they are crucial for POR activity [19]. De-tailed kinetic and structural studies are now necessary to fur-ther our understanding of the catalytic mechanism. However,an abundant source of pure enzyme is needed for this work.Our recent success overexpressing POR from pea in E. colias a fusion with maltose binding protein (MBP) and the sub-sequent demonstration of activity in vitro provided an excel-lent opportunity to study the structure and activity of thisenzyme [21]. More recently, this system has been used to over-express PORs from Synechocystis [22] and barley [23]. Themain drawback of this approach is that it has not yet beenpossible to cleave these fusion proteins and so all experimentshave been conducted with the 40 kDa MBP fused to the N-terminus of the enzyme. In the present paper, we report the