Kai-Hong Zhao

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the β-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the α-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

Chromophore Attachment to Phycobiliprotein β-Subunits PHYCOCYANOBILIN:CYSTEINE-β84 PHYCOBILIPROTEIN LYASE ACTIVITY OF CpeS-LIKE PROTEIN FROM ANABAENA Sp. PCC7120

The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-β84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-β84 of the apo-β-subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 °C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3μm, and a kcat = 1.7 × 10-5 and 1.1 × 10-5 s-1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS.

Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyase-isomerase.

Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters.

Fused‐Gene Approach to Photoswitchable and Fluorescent Biliproteins

Fluorescent and photoswitchable proteins are invaluable in life sciences and considered for applications in data storage. Of particular interest for in vivo studies are fluorescent proteins whose chromophores are generated autocatalytically from the amino acid chain; some of them can also be switched between two states. 3] Alternatively, apoproteins can be used that spontaneously incorporate endogenous chromophores like retinal. 5] The open-chain tetrapyrrole chromophore of biliproteins is subject to remarkable excited-state control of the chromophore by the apoprotein. Absorption and fluorescence of free bilins like the phycocyanobilin (PCB) is strongly increased in native biliproteins: the maximum can be shifted by over 100 nm, and a photochemical reaction path is opened in photochromic biliproteins like phytochromes and cyano(bacterio)chromes. These natural variations and the possibility to modulate the photophysical properties render biliproteins, in principle, excellent biomarkers and photonic materials. Applications have been limited, however, because the bilin chromophores must be provided separately and then attached covalently to the apoproteins. Previously, genes of the apoprotein were co-expressed with genes whose products generate the bilin chromophore from endogenous heme and then attach it covalently to the apoprotein. We now report an alternative approach that generates various biliproteins in situ from a single, multifunctional gene and endogenous heme. This approach is demonstrated by the synthesis of two persistently red-fluorescent biliproteins based on allophycocyanins, and by photochromic biliproteins derived from a novel cyanobacteriochrome that can be reversibly switched from a state absorbing and strongly fluorescing in the red, to a spectroscopically well-separated, less fluorescent state absorbing in the green spectral region. Gene slr1393 of the cyanobacterium Synechocystis sp. PCC6803 encodes a red–green photoreversible cyanobacteriochrome. The full-length protein contains three GAF domains, but GAF3 (aa 441–596) alone is capable of autocatalytically binding PCB to cysteine-528. Addition of PCB to GA results in a reversibly photochromic chromoprotein, termed RGS (red–green switchable protein): state Pr (lmax = 650 nm) is strongly fluorescent (FF = 0.06); it is reversibly converted by irradiation with red light into state Pg (lmax = 539 nm), which has reduced and strongly blueshifted fluorescence (Table 1, Figure 1a). Photoswitching can be repeated many times; it is stable over a wide pH range, and is retained after RGS is embedded into polyvinyl alcohol (PVA) film (see Figures S1 and S2 in the Supporting Information). Chromophorylated RGS can be produced in E. coli that has been multiply transformed to produce the GAF3 apoprotein and two biosynthetic enzymes generating PCB from heme, that is, heme oxygenase (HO1) and the biliverdin reductase (PcyA). The cells show an intense red fluorescence that can be abolished by irradiation with red light and is regained with green light (see Figure S2 in the Supporting Information). When pcyA was replaced by hy2, the phytochromobilin chromophore (PFB) was produced. The photochromic protein generated can be photoswitched reversibly between Pr (lmax = 663 nm) and Pg (lmax = 573 nm); in this case, both are moderately fluorescent (Table 1). HO1 and PcyA are thought to be involved in substrate channeling of the biliverdin produced by HO1; therefore, we fused the two genes and introduced the ho1:pcyA construct together with a plasmid containing the apoprotein gene, gaf3, into E. coli. These cells produced spectroscopically indistinguishable chromophorylated RGS in comparable yield (70–90%, Table 1) as previously with the separate plasmids. Finally, the gene gaf3 coding for the apoprotein was fused to ho1:pcyA at the 5’-end. E. coli cells expressing the [*] Dr. J. Zhang, X.-J. Wu, Z.-B. Wang, Y. Chen, Dipl.-Biol. X. Wang, Prof. M. Zhou, Prof. Dr. K.-H. Zhao State Key Laboratory of Agricultural Microbiology Huazhong Agricultural University Wuhan 430070 (P.R. China) Fax: (+ 86)27-8754-1634 E-mail: khzhao@163.com X.-J. Wu, Y. Chen, Dipl.-Biol. X. Wang, Prof. Dr. K.-H. Zhao College of Life Science and Technology Huazhong University of Science and Technology Wuhan 430074 (P.R. China)

Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores: photochemistry and dark reversion kinetics.

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full‐length, truncated and cysteine point‐mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV‐visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red–green photochromism (λmax = 650 and 535 nm) that is assigned to the reversible 15Z/E isomerization of a single phycocyanobilin‐chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E‐PCB was more active than the 650 nm state containing Z‐PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB‐GAF1 is photochromic, with the proposed 15E state (λmax = 685 nm) reverting slowly thermally to the thermostable 15Z state (λmax = 635 nm). PCB‐GAF3 showed a novel red–orange photochromism; the unstable state (putative 15E, λmax = 595 nm) reverts very rapidly (τ∼ 20 s) back to the thermostable Z state (λmax = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E‐GAF1/E‐GAF3 shows the highest rate of autophosphorylation activity, while E‐GAF1/Z‐GAF3 has intermediate activity, and Z‐GAF1/Z‐GAF3 is the least active state.

Lyase Activities of CpcS- and CpcT-like Proteins from Nostoc PCC7120 and Sequential Reconstitution of Binding Sites of Phycoerythrocyanin and Phycocyanin β-Subunits

Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647 (cpcT2) are homologous to the lyase cpcT. The functions of the encoded proteins were screened in vitro and in a heterologous Escherichia coli system with plasmids conferring biosynthesis of the phycocyanobilin chromophore and of the acceptor proteins β-phycoerythrocyanin (PecB) or β-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine β155 but does not discriminate between CpcB and PecB. The in vitro reconstitutions required no cofactors, and kinetic constants were determined for CpcT1 under in vitro conditions. No lyase activity was found for the lyase homologues CpcS2 and CpcT2, but complexes are formed in vitro between CpcT1 and CpcS1, CpcT2, or PecE (subunit of phycoviolobilin:α-phycoerythrocyanin isomerase lyase). The genes coding the inactive homologues, cpcS2 and cpcT2, are transcribed in N-starved Nostoc. In sequential binding experiments with CpcT1 and CpcS1, a chromophore at cysteine 84 inhibited the subsequent attachment to cysteine 155, whereas the inverse sequence generates subunits carrying both chromophores.

A rising tide of blue-absorbing biliprotein photoreceptors – characterization of seven such bilin-binding GAF domains in Nostoc sp. PCC7120

Cyanobacteriochromes are photochromic sensory photoreceptors in cyanobacteria that are related to phytochromes but cover a much broader spectral range. Using a homology search, a group of putative blue‐absorbing photoreceptors was identified in Nostoc sp. PCC 7120 that, in addition to the canonical chromophore‐binding cysteine of cyanobacteriochromes, have a conserved extra cysteine in a DXCF motif. To assess their photochemical activities, putative chromophore‐binding GAF domains were expressed in Escherichia coli together with the genes for phycocyanobilin biosynthesis. All except one covalently bound a chromophore and showed photoreversible photochromic responses, with absorption at approximately 420 nm for the 15Z states formed in the dark, and a variety of red‐shifted absorption peaks in the 490–600 nm range for the 15E states formed after light activation. Under denaturing conditions, the covalently bound chromophores were identified as phycocyanobilin, phycoviolobilin or mixtures of both. The canonical cysteines and those of the DXCF motifs were mutated, singly or together. The canonical cysteine is responsible for stable covalent attachment of the bilin to the apo‐protein at C31. The second linkage from the cysteine in the DXCF motif, probably to C10 of the chromophore, yields blue‐absorbing rubin‐type 15Z chromophores, but is lost in most cases upon photoconversion to the 15E isomers of the chromophores, and also when denatured with acidic urea.

The terminal phycobilisome emitter, LCM: A light-harvesting pigment with a phytochrome chromophore

Significance Photosynthesis, the basis for life on earth, relies on proper balancing of the beneficial and destructive potentials of light. In cyanobacteria and red algae, which contribute substantially to photosynthesis, the core-membrane linker, LCM, is critical to this process. Light energy harvested by large antenna complexes, phycobilisomes, is funneled to LCM. Depending on light conditions, LCM passes this energy productively to reaction centers that transform it into chemical energy or, on oversaturating conditions, to the photoprotecting orange carotenoid protein (OCP). The details of these functions in the complex-structured LCM are poorly understood. The crystal structure and time-resolved data of the chromophore domain of LCM provide a rationale for the functionally relevant energetic matching, and indicate a mechanism for switching between photoproductive and photoprotective functions. Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, LCM. The chromophore domain of LCM forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in LCM by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of LCM. Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.

Chromophore attachment in phycocyanin

Covalent attachment of phycocyanobilin (PCB) to the α‐subunit of C‐phycocyanin, CpcA, is catalysed by the heterodimeric PCB : CpcA lyase, CpcE/F [Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA & Glazer AN (1992) Proc Natl Acad Sci USA89, 7017–7021]. CpcE and CpcF of the cyanobacterium, Mastigocladus laminosus PCC 7603, form a 1 : 1 complex. Lyase‐mutants were constructed to probe functional domains. When in CpcE (276 residues) the N terminus was truncated beyond the R33YYAAWWL motif, or the C terminus beyond amino acid 237, the enzyme became inactive. Activity decreases to 20% when C‐terminal truncations went beyond L275, which is a key residue: the Km of CpcE(L275D) and (L276D) increased by 61% and 700%, kcat/Km decreased 3‐ and 83‐fold, respectively. The enzyme also lost activity when in CpcF (213 residues) the 20 N‐terminal amino acids were truncated; truncation of 53 C‐terminal amino acids inhibited complex formation with CpcE, possibly due to misfolding. According to chemical modifications, one accessible arginine and one accessible tryptophan are essential for CpcE activity, and one carboxylate for CpcF. Both subunits bind PCB, as assayed by Ni2+ affinity chromatography, SDS/PAGE and Zn2+‐induced fluorescence. The bound PCB could be transferred to CpcA to yield α‐CPC. The PCB transfer capacity correlates with the activity of the lyase, indicating that PCB bound to CpcE/F is an intermediate of the enzymatic reaction. A catalytic mechanism is proposed, in which a CpcE/F complex binds PCB and adjusts via a salt bridge the conformation of PCB, which is then transferred to CpcA.

Biliprotein Chromophore Attachment CHAPERONE-LIKE FUNCTION OF THE PecE SUBUNIT OFα-PHYCOERYTHROCYANIN LYASE

Biliproteins are post-translationally modified by chromophore addition. In phycoerythrocyanin, the heterodimeric lyase PecE/F covalently attaches phycocyanobilin (PCB) to cysteine-α84 of the apoprotein PecA, with concomitant isomerization to phycoviolobilin. We found that: (a) PecA adds autocatalytically PCB, yielding a low absorbance, low fluorescence PCB·PecA adduct, termed P645 according to its absorption maximum; (b) In the presence of PecE, a high absorbance, high fluorescence PCB·PecA adduct is formed, termed P641; (c) PecE is capable of transforming P645 to P641; (d) When in stop-flow experiments, PecA and PecE were preincubated before chromophore addition, a red-shifted intermediate (P650, τ = 32 ms) was observed followed by a second, which was blue-shifted (P605, τ = 0.5 s), and finally a third (P638, τ = 14 s) that yielded the adduct (P641, τ = 20 min); (e) The reaction was slower, and P605 was missing, if PecA and PecE were not preincubated; (f) Gel filtration gave no evidence of a stable complex between PecA and PecE; however, complex formation is induced by adding PCB; and (g) A red-shifted intermediate was also formed, but more slowly, with phycoerythrobilin, and denaturation showed that this is not yet covalently bound. We conclude, therefore, that PecA and PecE form a weak complex that is stabilized by PCB, that the first reaction step involves a conformational change and/or protonation of PCB, and that PecE has a chaperone-like function on the chromoprotein.