Purification, crystallization, NMR spectroscopy and biochemical analyses of alpha-phycoerythrocyanin peptides.

The alpha-phycoerythrocyanin subunits of the different phycoerythrocyanin complexes of the phycobilisomes from the cyanobacterium Mastigocladus laminosus perform a remarkable photochemistry. Similar to phytochromes - the photoreceptors of higher plants - the spectral properties of the molecule reversibly change according to the irradiation wavelength. To enable extensive analyses, the protein has been produced at high yield by improving purification protocols. As a result, several comparative studies on the Z- and E-configurations of the intact alpha-subunit, and also on photoactive peptides originating from nonspecific degradations of the chromoprotein, were possible. The analyses comprise absorbance, fluorescence and CD spectroscopy, crystallization, preliminary X-ray measurements, mass spectrometry, N-terminal amino acid sequencing and 1D NMR spectroscopy. Intact alpha-phycoerythrocyanin aggregates significantly, due to hydrophobic interactions between the two N-terminal helices. Removal of these helices reduces the aggregation but also destabilizes the protein fold. The complete subunit could be crystallized in its E-configuration, but the X-ray measurement conditions must be improved. Nevertheless, NMR spectroscopy on a soluble photoactive peptide presents the first insight into the complex chromophore protein interactions that are dependent on the light induced state. The chromophore environment in the Z-configuration is rigid whereas other regions of the protein are more flexible. In contrast, the E-configuration has a mobile chromophore, especially the pyrrole ring D, while other regions of the protein rigidified compared to the Z-configuration.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

Photochromic biliproteins from the cyanobacterium Anabaena sp. PCC 7120: lyase activities, chromophore exchange, and photochromism in phytochrome AphA

Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore. The cyanobacterium Anabaena sp. PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA). (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore. (b) AphA chromophorylation is autocatalytic such as in other phytochromes. (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC). The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor. (d) Through deletion mutations of aphA, a short sequence IQPHGV [amino acids (aa) 26-31] was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259. (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes. (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (相似文献   

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.     

Biochemical and spectroscopic characterization of the bacterial phytochrome of Pseudomonas aeruginosa

Phytochromes are photochromic biliproteins found in plants as well as in some cyanotrophic, photoautotrophic and heterotrophic bacteria. In many bacteria, their function is largely unknown. Here we describe the biochemical and spectroscopic characterization of recombinant bacterial phytochrome from the opportunistic pathogen Pseudomonas aeruginosa (PaBphP). The recombinant protein displays all the characteristic features of a bonafide phytochrome. In contrast with cyanobacteria and plants, the chromophore of this bacterial phytochrome is biliverdin IXalpha, which is produced by the heme oxygenase BphO in P. aeruginosa. This chromophore was shown to be covalently attached via its A-ring endo-vinyl group to a cysteine residue outside the defined bilin lyase domain of plant and cyanobacterial phytochromes. Site-directed mutagenesis identified Cys12 and His247 as being important for chromophore binding and photoreversibility, respectively. PaBphP is synthesized in the dark in the red-light-absorbing Pr form and immediately converted into a far-red-light-absorbing Pfr-enriched form. It shows the characteristic red/far-red-light-induced photoreversibility of phytochromes. A chromophore analog that lacks the C15/16 double bond was used to show that this photoreversibility is due to a 15Z/15E isomerization of the biliverdin chromophore. Autophosphorylation of PaBphP was demonstrated, confirming its role as a sensor kinase of a bacterial two-component signaling system.     

Assembly of synthetic locked chromophores with agrobacterium phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.     

The native forms of the phycobilin chromophores of algal biliproteins. A clarification.

Pigments released from phycoerythrins and phycocyanins by treatment with hot methanol are currently regarded as equivalent to the native chromophores phycoerythrobilin and phycocyanobilin. However, evidence presented here confirms the original view of O'Carra & O'hEocha [(1966 Phytochemistry 5, 993-997] that these methanol-released pigments are artefacts differing in their chromophoric conjugated systems from the native protein-bound prosthetic groups. By contrast, the native spectral properties are retained in pigments released by careful acid treatment of the biliproteins and these acid-released phycobilins, rather than the methanol-released pigments, are therefore regarded as the protein-free forms of the native chromophores. The conclusion reached by Chapman, Cole & Siegelman [(1968) J. Am. Chem. Soc. 89, 3643-3645], that all the algal biliproteins contain only phycoerythrobilin and phycocyanobilin, is shown to be incorrect. The identification of a urobilinoid chromophore, phycourobilin, accompanying phycoerythrobilin in B- and R- phycoerythrins is confirmed and supported by more extensive evidence. The cryptomonad phycocyanins are shown to contain a phycobilin chromophore accompanying phycocyanobilin. This further phycobilin has the spectral properties of the class of bilins known as violins and the provisional name "cryptoviolin" is proposed pending elucidation of its structure.     

Phytochrome signaling: solving the Gordian knot with microbial relatives

Phytochromes encompass a diverse collection of biliproteins that regulate numerous photoresponses in plants and microorganisms. Whereas the plant versions have proven experimentally intractable for structural studies, the microbial forms have recently provided important insights into how these photoreceptors work at the atomic level. Here, we review the current understanding of these microbial phytochromes, which shows that they have a modular dimeric architecture that propagates light-driven rotation of the bilin to distal contacts between adjacent signal output domains. Surprising features underpinning this signaling include: a deeply buried chromophore; a knot and hairpin loop that stabilizes the photosensing domain; and an extended helical spine that translates conformational changes in the photosensing domain to the output domain. Conservation within the superfamily both in modular construction and sequence strongly suggests that higher plant phytochromes work similarly as light-regulated toggle switches.     

Fluorescence of phytochrome adducts with synthetic locked chromophores

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended π-electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.     

Structural studies on cryptomonad biliprotein subunits. Two different alpha-subunits in Chroomonas phycocyanin-645 and Cryptomonas phycoerythrin-545

The N-terminal amino-acid sequences of two green alpha-subunit fractions from Chroomonas phycocyanin-645 and from two violet alpha-subunit fractions from Cryptomonas phycoerythrin-545 reveal that these cryptomonad biliproteins each contain two different alpha-subunits. The chromophore binding sites at the cysteine residues in positions 18 or 19 are homologous to the chromophore binding site at cysteine position 84 in cyanobacterial biliproteins. The sequence homologies of the beta-subunits to cyanobacterial biliproteins are higher than those of the alpha-subunits. Cryptomonas phycoerythrin-545 alpha-subunits contain a gamma-hydroxylysine residue at the fourth position of the polypeptide chains. 50%-75% of the total sequence of the alpha-subunits was determined by N-terminal amino-acid sequence analysis. The alpha-subunits of the Cryptomonad biliproteins are smaller than the alpha-subunits of the cyanobacterial biliproteins. Comparing sequence homologies we found 60 amino-acid residues less at the N-terminus of Cryptomonad biliproteins than in cyanobacterial biliproteins.     

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (alphabeta)3 resulted first in monomerization (alphabeta), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

Chromophore composition of the phycobiliprotein Cr-PC577 from the cryptophyte Hemiselmis pacifica

The cryptophyte phycocyanin Cr-PC577 from Hemiselmis pacifica is a close relative of Cr-PC612 found in Hemiselmis virescens and Hemiselmis tepida. The two biliproteins differ in that Cr-PC577 lacks the major peak at around 612 nm in the absorption spectrum. Cr-PC577 was thus purified and characterized with respect to its bilin chromophore composition. Like other cryptophyte phycobiliproteins, Cr-PC577 is an (αβ)(α′β) heterodimer with phycocyanobilin (PCB) bound to the α-subunits. While one chromophore of the β-subunit is also PCB, mass spectrometry identified an additional chromophore with a mass of 585 Da at position β-Cys-158. This mass can be attributed to either a dihydrobiliverdin (DHBV), mesobiliverdin (MBV), or bilin584 chromophore. The doubly linked bilin at position β-Cys-50 and β-Cys-61 could not be identified unequivocally but shares spectral features with DHBV. We found that Cr-PC577 possesses a novel chromophore composition with at least two different chromophores bound to the β-subunit. Overall, our data contribute to a better understanding of cryptophyte phycobiliproteins and furthermore raise the question on the biosynthetic pathway of cryptophyte chromophores.     

Phytochrome photoconversion

The spectral properties of native and modified phytochromes and the molecular events during phytochrome photoconversion, , are reviewed. Steady-state and time-resolved absorption spectra of native phytochrome A, as well as recombinant phytochromes (oat and potato phytochrome A and potato phytochrome B) reconstituted with phycocyanobilin and phytochromobilin as chromophores, are analysed. The vinyl double bond, present at position 18 in phytochromobilin and substituted by an ethyl group in phycocyanobilin, has a considerable influence on the photo-transformation kinetics of phytochromes A and B, evidently due to a strong interaction of this region of the chromophore with the protein surrounding. The kinetics of the phototransformation of potato phytochrome B differs from that of oat phytochrome A (wild-type and recombinant), indicating that the chromophore-protein interaction in phytochrome B is different from that in phytochrome A. It remains to be seen whether this difference is due to the di- versus monocotyledon origin of the phytochromes. Optoacoustic spectroscopy, applied to native oat phytochrome A, afforded thermo-dynamic, structural and kinetic parameters of the Pr→I₇₀₀ and the I₇₀₀→Pr phototransformations. Raman and infrared spectroscopic data for wild-type phytochrome A suggest that the protonated chromophore in Pr undergoes torsions around two single bonds in addition to the Z→E isomerization of the 15 ,16 double bond, and that all transients, possibly with the exception of I_bI, are protonated at the central pyrrole ring.     

Studies on plant bile pigments, VII. Preparation and characterization of phycobiliproteins with chromophores chemically modified by reduction.

The reversible denaturation and reduction with dithionite has been studied for the phycobiliproteins, C-phycocyanin (1) and allophycocyanin (2) from Spirulina platensis, and C-phycoerythrin (4) from Fremyella diplosiphon (both cyanobacteria). By treatment with sodium dithionite, the chromophores are selectively reduced at the central (C-10) methine bridge, producing pigments with bilirubinoid (lambda max = 418 nm from 1 and 2), and vinylpyrroloc (lambda max= 300 nm from 4) chromophores. The extent of reduction is dependent on the state of the protein. The chromophores of denatured biliproteins are completely reduced at 0.5 mM dithionite. In the native pigments, dithionite concentrations up to 0.5 mM lead only to partial reduction, thus forming products containing both reduced and oxidized chromophores (e.g. "phycocyanorubins" from 1 and 2). The reduction is non-statistical with respect to the different chromophores present in 1 and 4, the chromophores absorbing at shorter wavelengths being preferentially reduced. Renaturation of the proteins containing reduced chromophores is accompanied by their reoxidation. This oxidation is complete in the absence of dithionite or at concentrations up to 0.5 mM. At higher dithionite concentrations, the reoxidation is incomplete, and the products are spectroscopically identical to those obtained by reduction of the native pigments at similar concentrations of reductant. The results are interpreted by a model in which the protein is "transparent" to the reducing agent, dithionite. The difference in the extent of reduction of the native and denatured pigments can only be due to thermodynamic (viz. stability) differences in the susceptibility of the chromophores to reduction. Specifically, the (extended) chromophore present in the native pigment is much more difficult to reduce than the chromophore (present in a cyclic conformation) in the denatured pigment. The energetics of the process of refolding both the protein and the chromophores are discussed.     

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions: 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (αβ)₃ resulted first in monomerization (αβ), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXalpha. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45A resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C3(2) carbon of biliverdin to Cys(24), the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors.     

A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/ E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.     

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.     

A New Appraisal of the Prokaryotic Origin of Eukaryotic Phytochromes

The evolutionary origin of the phytochromes of eukaryotes is controversial. Three cyanobacterial proteins have been described as ``phytochrome-like' and have been suggested to be potential ancestors of these essential photoreceptors: Cph1 from Synechocystis PCC 6803, showing homology to phytochromes along its entire length and known to attach a chromophore; and PlpA from Synechocystis PCC 6803 and RcaE from Fremyella diplosiphon, both showing homology to phytochromes most strongly only in the C-terminal region and not known to bind a chromophore. We have reexamined the evolution of the photoreceptors using for PCR amplification a highly conserved region encoding the chromophore-binding domain in both Cph1 and phytochromes of plants and have identified genes for phytochrome-like proteins (PLP) in 11 very diverse cyanobacteria. The predicted gene products contain either a Cys, Arg, Ile, or Leu residue at the putative chromophore binding site. In 10 of the strains examined only a single gene was found, but in Calothrix PCC 7601 two genes (cphA and cphB) were identified. Phylogenetic analysis revealed that genes encoding PLP are homologues that share a common ancestor with the phytochromes of eukaryotes and diverged before the latter. In contrast, the putative sensory/regulatory proteins, including PlpA and RcaE, that lack a part of the chromophore lyase domain essential for chromophore attachment on the apophytochrome, are only distantly related to phytochromes. The Ppr protein of the anoxygenic photosynthetic bacterium Rhodospirillum centenum and the bacterial phytochrome-like proteins (BphP) of Deinococcus radiodurans and Pseudomonas aeruginosa fall within the cluster of cyanobacterial phytochromes. Received: 9 December 1999 / Accepted: 10 May 2000