Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.     

Substrate-specificity studies on protochlorophyllide reductase in barley (Hordeum vulgare) etioplast membranes.

1. The substrate specificity of the enzyme protochlorophyllide reductase in barley (Hordeum vulgare) etioplasts was investigated. 2. It was shown that naturally occurring esterified protochlorophyllide and chemically prepared protochlorophyllide methyl ester are not substrates for the enzyme, suggesting an important role for the C-7 carboxylic acid group in binding of the porphyrin to the enzyme. 3. Removal of magnesium from the protochlorophyllide leads to inactivity of the compound as a substrate for the enzyme. However, activity can be restored by replacing the magnesium with zinc, whereas nickel, copper or cobalt failed to restore substrate activity. 4. Binding of the second substrate, NADPH, to the enzyme probably occurs through the 2'-phosphate group in the coenzyme.     

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.     

Photoactive pigment—enzyme complexes of chlorophyll precursor in plant leaves

This review summarizes contemporary data on structure and function of photoactive pigment--enzyme complexes of the chlorophyll precursor that undergoes photochemical transformation to chlorophyllide. The properties and functions of the complex and its principal components are considered including the pigment (protochlorophyllide), the hydrogen donor (NADPH), and the photoenzyme protochlorophyllide oxidoreductase (POR) that catalyzes the photochemical production of chlorophyllide. Chemical variants of the chlorophyll precursor are described (protochlorophyllide, protochlorophyll, and their mono- and divinyl forms). The nature and photochemical activity of spectrally distinct native protochlorophyllide forms are discussed. Data are presented on structural organization of the photoenzyme POR, its substrate specificity, localization in etioplasts, and heterogeneity. The significance of different POR forms (PORA, PORB, and PORC) in adaptation of chlorophyll biosynthesis to various illumination conditions is considered. Attention is paid to structural and functional interactions of three main constituents of the photoactive complex and to possible existence of additional components associated with the pigment-enzyme complex. Historical aspects of the problem and the prospects of further investigations are outlined.     

Enzymes of the last steps of chlorophyll biosynthesis: modification of the substrate structure helps to understand the topology of the active centers

Enzymes catalyzing two of the late steps of chlorophyll biosynthesis are NADPH:protochlorophyllide oxidoreductase (POR), responsible for the light-dependent reduction of protochlorophyllide to chlorophyllide, and chlorophyll synthase that catalyses the esterification of chlorophyllide to chlorophyll. Inhibitors of these enzymes are of interest as potential herbicides. Both enzymes presumably form a complex, and the question arose whether chlorophyll synthase can react with chlorophyllide while it is still bound to POR. Here, we describe the chemical modification of protochlorophyllides and chlorophyllides with space-filling substituents at rings A, B, and E of the tetrapyrrole macrocycle and the reactivity of the modified substrates. Both enzymes tolerate the large and flexible phenylamino substituent at ring B, indicating that ring B points toward the enzyme surface while the substrate is bound. On the basis of the standard compound zinc protopheophorbide a (100% activity), the 7(1)-phenylamino derivative shows a comparable activity (83%) with POR that is higher than that of the parent formyl derivative zinc protopheophorbide b (58% activity). In contrast, the 3(1)-phenylamino derivative is less active (12%) than the parent formyl compound zinc protopheophorbide d (49% activity), indicating that the binding pocket leaves less space around ring A than around ring B. Almost no space must be left around ring E because substitution of the 13(2)-carboxymethyl ester (100% activity) by the 13(2)-carboxyethyl ester reduces the activity to 0.2%. Chlorophyll synthase leaves somewhat more space around ring E on the A side of the tetrapyrrole in the binding pocket; substitution of the 13(2)-proton (100% activity) by a methoxy group (53% activity) and an ethoxy group (11% activity) is tolerated to a certain extent, while the carbomethoxy group in this position is not accepted. Opening of ring E to a chlorin e6 dimethylester is tolerated (39% activity), while the large benzylamide residue at this site leads to the loss of activity. We conclude that the tetrapyrroles bind to both enzymes in the same direction: rings C, D, and E are oriented to the interior of the binding cleft, and rings A and B are oriented to the surface of the enzyme; this excludes simultaneous binding to both enzymes.     

Substrate recognition of nitrogenase-like dark operative protochlorophyllide oxidoreductase from Prochlorococcus marinus

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL(2) dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)(2) complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)(2). For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.     

POR hits the road: import and assembly of a plastid protein

The biosynthesis of chlorophyll is a strictly light-dependent multistep process in higher plants. The light-dependent step is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1), which reduces protochlorophyllide (Pchlide) to chlorophyllide (Chlide). POR is nucleus-encoded and post-translationally imported into plastids. It has been proposed that the import of a POR protein isozyme (PORA) is totally dependent on Pchlide and uses a novel import pathway. This proposal is based on findings that PORA import only occurs in the presence of Pchlide and that the presence of overexpressed precursor of Rubisco small subunit (pSS), a protein which is known to use the general import pathway, does not outcompete PORA import. Another study demonstrated that POR precursor protein (pPOR) can be cross-linked to one of the components in the translocation machinery, Toc75, in the absence of Pchlide, and that its import can be outcompeted by the addition of the pSS. This indicates that pSS and pPOR may use the same translocation mechanism. Thus, POR does not necessarily need Pchlide for import – which is in contrast to earlier observations – and the exact POR import mechanism remains unresolved. Once in the stroma, the POR transit peptide is cleaved off and the mature POR protein is associated to the plastid inner membranes. Formation of the correct membrane–associated, thermolysin-protected assembly is strictly dependent of NADPH. As a final step, the formation of the NADPH-Pchlide-POR complex occurs. When POR accumulates in the membranes of proplastids, an attraction of monogalactosyl diacylglycerol (MGDG) can occur, leading to the formation of prolamellar bodies (PLBs) and the development of etioplasts in darkness.     

Etioplast differentiation in arabidopsis: both PORA and PORB restore the prolamellar body and photoactive protochlorophyllide-F655 to the cop1 photomorphogenic mutant.

The etioplast plastid type of dark-grown angiosperms is defined by the accumulation of the chlorophyll (Chl) precursor protochlorophyllide (Pchlide) and the presence of the paracrystalline prolamellar body (PLB) membrane. Both features correlate with the presence of NADPH:Pchlide oxidoreductase (POR), a light-dependent enzyme that reduces photoactive Pchlide-F655 to chlorophyllide and plays a key role in chloroplast differentiation during greening. Two differentially expressed and regulated POR enzymes, PORA and PORB, have recently been discovered in angiosperms. To investigate the hypothesis that etioplast differentiation requires PORA, we have constitutively overexpressed PORA and PORB in the Arabidopsis wild type and in the constitutive photomorphogenic cop1-18 (previously det340) mutant, which is deficient in the PLB and Pchlide-F655. In both genetic backgrounds, POR overexpression increased PLB size, the ratio of Pchlide-F655 to nonphotoactive Pchl[ide]-F632, and the amount of Pchlide-F655. Dramatically, restoration of either PORA or PORB to the cop1 mutant led to the formation of etioplasts containing an extensive PLB and large amounts of photoactive Pchlide-F655.     

Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH: protochlorophyllide oxidoreductase from Synechocystis

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes. A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the Synechocystis POR enzyme. The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni(2+)-affinity column. The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity. The K(d) value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced.     

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase

Using circular dichroism (CD) spectroscopy, the stereochemistry at C-13(2) of members of the chlorophyll (Chl) c family, namely Chls c(1), c(2), c(3) and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c(1) is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-13(2), which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c(1) is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates.     

POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.     

光下叶绿素缺乏的大麦突变体NYB中叶绿素合成再探

研究一种原叶绿素酯还原酶（POR）的大麦突变体Ⅳ阳光下合成叶绿素的结果表明,NYB中PORB的含量比野生型低。NYB前质体中原片层体的大小和数量与野生型差不多,但其结构比野生型的松散。暗中生长的突变体内POR蛋白复合物LHPP比野生型少。不同光照强度下叶绿素积累的结果显示,光照度越强,突变体与野生型的叶绿素差异越显著。由于porB是单拷贝的,所以推测突变体中部分porB mRNA可能产生错误的剪切拼接,以致光下突变体Ⅳ阳仍然能合成叶绿素。     

Two NADPH:Protochlorophyllide Oxidoreductases in Barley: Evidence for the Selective Disappearance of PORA during the Light-Induced Greening of Etiolated Seedlings

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.     

Prolamellar bodies formed by cyanobacterial protochlorophyllide oxidoreductase in Arabidopsis

In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto‐plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.     

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

Kinetic characterisation of the light-driven protochlorophyllide oxidoreductase (POR) from Thermosynechococcus elongatus.

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of the C17-C18 double bond of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), which is a key regulatory step in the chlorophyll biosynthesis pathway. POR from the thermophilic cyanobacterium Thermosynechococcus elongatus is an attractive system for following the reaction and in the present work we have carried out a detailed steady state kinetic characterisation of this enzyme. The thermophilic POR was shown to have maximal activity at approximately 50 degrees C, which is similar to the growth temperature of the organism. The V(max) was calculated to be 0.53 microM min(-1) and the K(m) values for NADPH and Pchlide were 0.013 microM and 1.8 microM, respectively. The binding properties for both substrates as well as the NADP(+) product have been analysed by using fluorescence emission measurements, which have allowed the dissociation constants for binding to be calculated. These results represent the first steady state kinetic characterisation of a thermophilic version of POR.