The specificity of the bilin lyase CpcS for chromophore attachment to allophycocyanin in the chlorophyll f-containing cyanobacterium Halomicronima hongdechloris

Phycobilisomes are light-harvesting antenna complexes of cyanobacteria and red algae that are comprised of chromoproteins called phycobiliproteins. PBS core structures are made up of allophycocyanin subunits. Halomicronema hongdechloris (H. hongdechloris) is one of the cyanobacteria that produce chlorophyll f (Chl f) under far-red light and is regulated by the Far-Red Light Photoacclimation gene cluster. There are five genes encoding APC in this specific gene cluster, and they are responsible for assembling the red-shifted PBS in H. hongdechloris grown under far-red light. In this study, the five apc genes located in the FaRLiP gene cluster were heterologously expressed in an Escherichia coli reconstitution system. The canonical APC-encoding genes were also constructed in the same system for comparison. Additionally, five annotated phycobiliprotein lyase-encoding genes (cpcS) from the H. hongdechloris genome were phylogenetically classified and experimentally tested for their catalytic properties including their contribution to the shifted absorption of PBS. Through analysis of recombinant proteins, we determined that the heterodimer of CpcS-I and CpcU are able to ligate a chromophore to the APC-α/APC-β subunits. We discuss some hypotheses towards understanding the roles of the specialised APC and contributions of PBP lyases.

     

Characterization of the bilin attachment sites in R-phycoerythrin

The amino acid sequence around the sites of attachment of all the bilin prosthetic groups of Gastroclonium coulteri R-phycoerythrin, (alpha beta)6 gamma, have been determined. The sequences of tryptic peptides derived from the alpha and beta subunits are (Formula: see text) where the designations alpha and beta refer to the subunits from which the peptides derived. Cysteinyl residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB). Spectroscopic studies on the gamma subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, gamma-A through gamma-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (alpha beta)6 gamma and (alpha beta)6 gamma', with gamma subunits differing in amino acid sequence. The sequences of the gamma subunit bilin peptides (see below) were not homologous to those from alpha and beta subunits of any biliprotein. (Formula: see text) The bilins in all these peptides are attached through single linkages to a cysteinyl residue, except for the phycourobilin on peptide beta-3 which is attached through two thioether linkages to cysteinyl residues 10 amino acids apart. The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/alpha beta in each of these three biliprotein types.     

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in <Emphasis Type=Italic>Escherichia coli</Emphasis>

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC α and β subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.     

Fluorescence properties of allophycocyanin and a crosslinked allophycocyanin trimer

Data on the wavelength and temperature dependence of both time-resolved and steady state fluorescence emission are presented for allophycocyanin (AP) and for a crosslinked allophycocyanin trimer (XL-AP) (Ong LJ and Glazer AN: Physiol Veg 23:777-787, 1985). AP dissociates at high dilution and is not stable above 40 degrees C even at moderate protein concentration. In contrast, XL-AP does not dissociate even at very low protein concentrations and is completely stable up to 60 degrees C in the presence of 0.75 M NaK-phosphate, pH 7.0. The results show that XL-AP is superior to AP for use in conjugates that absorb and emit in the red region of the spectrum. The high stability of XL-AP at elevated temperatures at high phosphate concentrations suggests that this derivative may be useful in conjunction with nucleic acid probes.     

Posttranslational modifications of the beta subunit of a cryptomonad phycoerythrin. Sites of bilin attachment and asparagine methylation

Determination of the partial amino acid sequence of the beta subunit of cryptomonad strain CBD phycoerythrin 566 established the nature, locations, and modes of attachment of the three bilin prosthetic groups and revealed a site of posttranslational methylation. Isolation of peptides cross-linked by a phycobiliviolin led to an unambiguous assignment of two thioether linkages, from residues beta-Cys-50 and beta-Cys-61 to this bilin. Two bilins were attached through single thioether linkages, a phycobiliviolin at beta-Cys-158 and a phycoerythrobilin at beta-Cys-82 (the residue numbering is that for B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Morisset, W., Wehrmeyer, W., and Zuber, H. (1985) Biol. Chem. Hoppe-Seyler 366, 233-244). The partial sequences (99 residues) established for phycoerythrin 566 beta subunit showed a 79% identity with that of the red algal Porphyridium cruentum B-phycoerythrin beta subunit. A particularly remarkable finding is that the unique methylasparagine residue at position beta-72, highly conserved in cyanobacterial and red algal phycobiliproteins (Klotz, A. V., and Glazer, A. N. (1987) J. Biol. Chem. 262, 17350-17355), is also present at beta-72 in the cryptomonad phycoerythrin. Comparison of the locations of donor and acceptor bilins in cryptomonad phycoerythrin with those found for cyanobacterial and red algal phycobiliproteins showed different favored pathways of energy migration in the cryptomonad protein.     

Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment.

Mutants of the cyanobacterium Synechococcus sp. PCC 7002 constructed by the insertional inactivation of either the cpcE or cpcF gene produce low levels of spectroscopically detectable phycocyanin. The majority of the phycocyanin produced in these strains appears to lack the alpha subunit phycocyanobilin (PCB) chromophore (Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R., and Bryant, D. A. (1992) J. Biol. Chem. 267, 16138-16145). Purification of the phycocyanin produced in the mutants revealed two fractions each with an aberrant absorption spectrum. Tryptic peptide maps of the major fraction showed that the alpha-84 PCB peptide was absent. The two PCB peptides derived from the beta subunit were normal. Tryptic digests of the less abundant phycocyanin fraction contained a family of bilin peptides derived from the alpha subunit. Several distinct bilin adducts were present. A major component was a mesobiliverdin adduct, a previously described product of the in vitro reaction of PCB and apophycocyanin. The same results were obtained with both the cpcE mutant and the cpcF mutant. In vitro reactions with PCB and the fractions containing apo alpha subunit showed that the alpha-84 bilin attachment site was unmodified and competent for adduct formation. Pseudo-revertants of both strains were observed to arise at high frequency. Analysis of the phycocyanin from a cpcE pseudo-revertant, which produced a near wild-type level of phycocyanin with alpha subunit carrying PCB, revealed a single amino acid substitution, alpha-Tyr129----Cys. This residue, which is conserved in all phycocyanins sequenced to date, forms part of the alpha-84 bilin binding site and lies within 5 A of alpha-Cys84. A mutated cpcA gene containing this substitution was constructed by site-directed mutagenesis and transformed, along with cpcB, into a cpcBAC deletion strain containing an insertionally inactivated cpcE. This strain produces high levels of phycocyanin and the majority of the alpha subunit carries PCB at alpha-Cys84.     

Kinetics of phycocyanine bilin groups destruction by peroxyl radicals

Bilin groups in c-phycocyanine are readily bleached by peroxyl radicals produced in the thermolysis of 2, 2'-azobis(2-amidinopropane). From an evaluation of the bilin groups destroyed per radical that interacts with the protein, it is concluded that the bilin moiety is the main target of the radicals. Kinetic expressions are derived that allows an estimation of the substrate reactivity from the analysis of the rate of bilin group modification as a function of the protein concentration. From this analysis it is concluded that micromolar concentrations of c-phycocyanine are able to reduce the steady state concentration of the peroxyl radicals by one half, indicating a high antioxidant activity for this compound. This conclusion is confirmed by measuring the capacity of the protein to protect 1-naphthol from modification by peroxyl radicals. The results obtained show that the bilin groups have, on a molar basis, an antioxidant activity similar to that of potent antioxidants such as catechin.     

Membrane attachment of recombinant G-protein alpha-subunits in excess of beta gamma subunits in a eukaryotic expression system

Recombinant cDNAs encoding the alpha-subunits of Gi1, Gi2, Gi3, Go and Gs were transfected into COS cells with the pCD-PS mammalian expression vector. Expression of each G alpha was verified using subtype-specific peptide antisera on immunoblots. Quantitative immunoblotting of alpha and beta subunits indicated: i) that there was no change in expression of endogenous beta subunits, and ii) overexpression of alpha subunits could achieve a ratio of alpha:beta greater than 25:1. Despite the excess of alpha over beta, the G alpha subunits were found predominantly in the membrane fraction. The results demonstrate that G alpha subunits can attach to the membrane independently of beta gamma subunits.     

Asp-225 and glu-375 in autocatalytic attachment of the prosthetic heme group of lactoperoxidase.

The heme in lactoperoxidase is attached to the protein by ester bonds between the heme 1- and 5-methyl groups and Glu-375 and Asp-275, respectively. To investigate the cross-linking process, we have examined the D225E, E375D, and D225E/E375D mutants of bovine lactoperoxidase. The heme in the E375D mutant is only partially covalently bound, but exposure to H(2)O(2) results in complete covalent binding and a fully active protein. Digestion of this mutant shows that the heme is primarily bound through its 5-methyl group. Excess H(2)O(2) increases the proportion of the doubly linked species without augmenting enzyme activity. The D225E mutant has little covalently bound heme and a much lower activity, neither of which are significantly increased by the addition of heme and H(2)O(2). The heme is linked to this protein through a single bond to the 1-methyl group. The D225E/E375D mutant has no covalently bound heme and no activity. A small amount of iron 1-hydroxymethylprotoporphyrin IX is obtained from the wild-type enzyme along with the predominant dihydroxylated derivative. The results establish that a single covalent link suffices to achieve maximum catalytic activity and suggest that the 5-hydroxymethyl bond may form before the 1-hydroxymethyl bond.     

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.     

Synthesis of fluorescent oligosaccharides for covalent attachment to living cells

Asparagine-linked oligosaccharides were liberated from glycoproteins by hydrazinolysis. The treatment resulted in de-N-acetylation of the amino sugars. After isolation of the oligosaccharides free amino groups were labeled with fluorescein isothiocyanate and remaining amino groups reacetylated. The fluorescent oligosaccharides were used to label living cells. They were converted to hydrazine derivatives and covalently attached to cell surface oligosaccharides, which had been treated with periodate or neuraminidase and galactose oxidase. This enabled the visualization of the attached oligosaccharides at the external aspect of the plasma membrane by fluorescence microscopy.     

Chromophores of allophycocyanin and R-phycocyanin

The biliprotein allophycocyanin was purified from Phormidium luridum, Anabaena variabilis and Plectonema boryanum. R-phycocyanin was purified from Rhodymenia palmata. The chromophores were cleaved from the denatured protein by methanol hydrolysis. They were purified and crystallized as the dimethyl esters. Chromatographic and absorption-spectral (visible–ultraviolet and infrared) comparisons with reference material have established phycocyanobilin as the chromophore of allophycocyanin. Phycocyanobilin and phycoerythrobilin were shown to be the chromophores of R-phycocyanin.     

Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin beta-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin beta-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin beta-subunit.     

In vitro attachment of bilins to apophycocyanin. II. Determination of the structures of tryptic bilin peptides derived from the phycocyanobilin adduct

In vitro reaction of phycocyanobilin (PCB) with apophycocyanin results in the specific addition of the bilin to two of the cysteinyl residues, alpha-Cys-84 and beta-Cys-82, which normally function in PCB attachment (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). These bilin binding sites are designated alpha-1 and beta-1, respectively. Tryptic digestion of the apophycocyanin-PCB adduct releases two major bilin peptides, alpha-1 mesobiliverdin (MBV) and beta-1 MBV, which encompass the two bilin-binding sites. These peptides were examined by 1H NMR and fast atom bombardment mass spectroscopies. The NMR spectra show that the bilin is attached to each peptide through a thioether linkage identical to the linkage observed in the corresponding tryptic peptides, alpha-1 PCB and beta-1 PCB, derived from the natural product, C-phycocyanin. However, the NMR spectra of the adduct peptides lack the resonances corresponding to protons at positions C2 and C3 of ring A seen in the spectra of the alpha-1 PCB and beta-1 PCB peptides. Fast atom bombardment mass spectroscopy shows the masses of the alpha-1 MBV and beta-1 MBV peptides to be 2 atomic mass units lower than those of the alpha-1 PCB and beta-1 PCB peptides, respectively. Comparison of the bilin portion of the NMR spectra of the alpha-1 MBV and beta-1 MBV peptides to the NMR spectra of PCB and mesobiliverdin confirms that the bilin of the two adduct peptides resembles mesobiliverdin in having an extra double bond in the C2-C3 position of ring A. These results show that the major bilin products arising from the reaction of PCB with apophycocyanin differ from the bilins present in C-phycocyanin. The relevance of these results to the biosynthetic pathway for the attachment of tetrapyrroles to phycobiliproteins is discussed.     

An unusual choanoflagellate protein released by Hedgehog autocatalytic processing

Hedgehog proteins are important cell-cell signalling proteins utilized during the development of multicellular animals. Members of the hedgehog gene family have not been detected outside the Metazoa, raising unanswered questions about their evolutionary origin. Here we report a highly unusual hedgehog-related gene from a choanoflagellate, a close unicellular relative of the animals. The deduced C-terminal domain, Hoglet-C, is homologous to the autocatalytic domain of Hedgehog proteins and is predicted to function in autocatalytic cleavage of the precursor peptide. In contrast, the N-terminal Hoglet-N peptide has no similarity to the signalling peptide of Hedgehog (Hh-N). Instead, Hoglet-N is deduced to be a secreted protein with an enormous threonine-rich domain of unprecedented size and purity (over 200 threonine residues) and two polysaccharide-binding domains. Structural modelling reveals that these domains have a novel combination of features found in cellulose-binding domains (CBD) of types IIa and IIb, and are expected to bind cellulose. We propose that the two CBD domains enable Hoglet-N to bind to plant matter, tethering an amorphous nucleophilic anchor, facilitating transient adhesion of the choanoflagellate cell. Since Hh-C and Hoglet-C are homologous, but Hh-N and Hoglet-N are not, we argue that metazoan hedgehog genes evolved by fusion of two distinct genes.     

Surface-enhanced resonance Raman spectroscopy of phycocyanin and allophycocyanin

High quality surface-enhanced resonance Raman (SERR) spectra were recorded from native and denatured phycocyanin and allophycocyanin on ascorbic acid treated silver hydrosols. The visible-excited SERR and resonance Raman (RR) spectra of the phycobiliproteins were very similar, indicating a predominantly electromagnetic surface enhancement mechanism. Investigation of pH-induced denaturation ofx allophycocyanin has shown that even small differences in protein/chromophore conformational are sensitively reflected by the SERR spectra. Concerning the adsorption of the protein to the metal surface, the experiments have shown that: (i) there is limited possibility for changing protein conformation during the adsorption process, (ii) there are no changes after the protein has been adsorbed onto the silver surface and (iii) for each protein an optimal activation of the silver sol has to be found for recording proper SERR spectra. The results obtained on phycobiliproteins are also discussed in connection with the interpretation of phytochrome Raman spectra.     

Biogenesis of phycobiliproteins: II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 OF beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002

The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcS-I copurified with His(6)-tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apophycocyanin, the reaction product had an absorbance maximum of 622 nm and was highly fluorescent (lambda(max) = 643 nm). In control reactions with PCB and apophycocyanin, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). Tryptic peptide mapping showed that the CpcSU-dependent reaction product had one major PCB-containing peptide that contained the PCB binding site Cys-82. The CpcSU lyase was also tested with recombinant apoHT-allophycocyanin (aporHT-AP) and PCB in vitro. AporHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of approximately 27 kDa. When aporHT-AP was incubated with PCB and CpcSU, the product had an absorbance maximum of 614 nm and a fluorescence emission maximum at 636 nm, the expected maxima for monomeric holo-AP. When no enzyme or CpcS-I or CpcU was added alone, the products had absorbance maxima between 645 and 647 nm and were not fluorescent. When these reaction products were analyzed by gel electrophoresis and zinc-enhanced fluorescence emission, only the reaction products from CpcSU had PCB attached to both AP subunits. Therefore, CpcSU is the bilin lyase-responsible for attachment of PCB to Cys-82 of CpcB and Cys-81 of ApcA and ApcB.     

A model of autocatalytic replication

Summary The catalytic effects that existing polymer chains have on the formation of new chains are modeled using ideas from spin glasses and neural networks. Computer simulation shows that isolated groups of chains in this model are capable of accurately replicating a wide variety of complex structures without templating. Replication in the model arises spontaneously and rapidly, leading to an extremely simple realization of a system exhibiting Darwinian evolution.     

Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism

Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal beta barrel ('beta domain') that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp(1120)) and an asparagine (Asn(1023)) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp(1120) and Asn(1023) form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.