A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.

The Hydrolysis of Pigment-Protein Phycoerythrin by Bromelain Enhances the Color Stability.

Phycoerythrin (PE) is a natural protein-pigment complex with a strong pink color, but it is sensitive to thermal and light variations. In this study, PE was extracted from Porphyra haitanensis in a yield of 0.2% (w/w). The phycoerythrin hydrolysates (PEH) (3-10 kDa) were prepared by enzymatic hydrolysis of PE with bromelain (8000 U/g) at 47 °C for 30 min, with a degree of hydrolysis (DH) of 11.57 ± 0.39% and a color degradation rate of 7.98 ± 0.39%. The physicochemical properties of PEH were evaluated. The UV and fluorescence spectra indicated that bromelain changed the microenvironment around phycoerythrobilin (PEB). The infrared spectrum revealed that the bromelain hydrolysis increased the α-helix content of PEH. The scanning electron microscope showed that bromelain destroyed the dense and smooth structure of PE, resulting in irregular porous structures. The radical scavenging activities of DPPH and ABTS of PEH were increased relative to that of PE (p < 0.05). The thermal (50-80 °C)-, UV (0.5-3 h)-, visible light irradiation (2-8 h)-, and metal ion exposing stabilities of PEH were significantly improved (p < 0.05). This study provides a potential scheme for overcoming the sensitivity of PE to thermal and light variations and facilitates PEH as a natural colorant ingredient in food and pigment applications.

Dichromic Allophycocyanin Trimer Covering a Broad Spectral Range (550-660†nm).

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, are a resource for photosynthetic, photonic and fluorescence labeling elements. They cover an exceptionally broad spectral range, but the complex superstructure and assembly have been an obstacle. By replacing in Synechocystis sp. PCC 6803 the biliverdin reductases, we studied the role of chromophores in the assembly of the phycobilisome core. Introduction of the green-absorbing phycoerythrobilin instead of the red-absorbing phycocyanobilin inhibited aggregation. A novel, trimeric allophycocyanin (Dic-APC) was obtained. In the small (110 kDa) unit, the two chromophores, phycoerythrobilin and phytochromobilin, cover a wide spectral range (550 to 660 nm). Due to efficient energy transfer, it provides an efficient artificial light-harvesting element. Dic-APC was generated in vitro by using the contained core-linker, LC , for template-assisted purification and assembly. Labeling the linker provides a method for targeting Dic-APC.

The phycoerythrobilin isomerization activity of MpeV in Synechococcus sp. WH8020 is prevented by the presence of a histidine at position 141 within its phycoerythrin-I ß-subunit substrate.

Marine Synechococcus efficiently harvest available light for photosynthesis using complex antenna systems, called phycobilisomes, composed of an allophycocyanin core surrounded by rods, which in the open ocean are always constituted of phycocyanin and two phycoerythrin (PE) types: PEI and PEII. These cyanobacteria display a wide pigment diversity primarily resulting from differences in the ratio of the two chromophores bound to PEs, the green-light absorbing phycoerythrobilin and the blue-light absorbing phycourobilin. Prior to phycobiliprotein assembly, bilin lyases post-translationally catalyze the ligation of phycoerythrobilin to conserved cysteine residues on α- or ß-subunits, whereas the closely related lyase-isomerases isomerize phycoerythrobilin to phycourobilin during the attachment reaction. MpeV was recently shown in Synechococcus sp. RS9916 to be a lyase-isomerase which doubly links phycourobilin to two cysteine residues (C50 and C61; hereafter C50, 61) on the ß-subunit of both PEI and PEII. Here we show that Synechococcus sp. WH8020, which belongs to the same pigment type as RS9916, contains MpeV that demonstrates lyase-isomerase activity on the PEII ß-subunit but only lyase activity on the PEI ß-subunit. We also demonstrate that occurrence of a histidine at position 141 of the PEI ß-subunit from WH8020, instead of a leucine in its counterpart from RS9916, prevents the isomerization activity by WH8020 MpeV, showing for the first time that both the substrate and the enzyme play a role in the isomerization reaction. We propose a structural-based mechanism for the role of H141 in blocking isomerization. More generally, the knowledge of the amino acid present at position 141 of the ß-subunits may be used to predict which phycobilin is bound at C50, 61 of both PEI and PEII from marine Synechococcus strains.

Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity.

Cryptophytes are among the few eukaryotes employing phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to cyanobacterial PBP that are organized in membrane-associated phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin 545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. The assembly of PBPs in cryptophytes involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of the eukaryotic S-type PBP lyase GtCPES. We show GtCPES-mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP ß-subunit (PmCpeB) of Prochlorococcus marinus MED4. On the basis of the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB), most likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colored complex in vitro and produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on these findings, we postulate that this single amino acid residue has a crucial role for bilin binding specificity of S-type phycoerythrin lyases but additional factors regulate handover to the target protein.

Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions.

Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from Spirulina subsalsa, covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λabs.max = 520 nm, λfluor.max = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.

MpeV is a lyase isomerase that ligates a doubly linked phycourobilin on the ß-subunit of phycoerythrin I and II in marine Synechococcus.

Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the ß-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into ß-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.

CpeT is the phycoerythrobilin lyase for Cys-165 on ß-phycoerythrin from Fremyella diplosiphon and the chaperone-like protein CpeZ greatly improves its activity.

Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae. The lyases responsible for chromophorylating the light harvesting protein phycoerythrin (PE) have not been fully characterized. In this study, we explore the role of CpeT, a putative bilin lyase, in the biosynthesis of PE in the cyanobacterium Fremyella diplosiphon. Recombinant protein studies show that CpeT alone can bind phycoerythrobilin (PEB), but CpeZ, a chaperone-like protein, is needed in order to correctly and efficiently attach PEB to the ß-subunit of PE. MS analyses of the recombinant ß-subunit of PE coexpressed with CpeT and CpeZ show that PEB is attached at Cys-165. Purified phycobilisomes from a cpeT knockout mutant and wild type (WT) samples from F. diplosiphon were analyzed and compared. The cpeT mutant contained much less PE and more phycocyanin than WT cells grown under green light, conditions which should maximize the production of PE. In addition, Northern blot analyses showed that the cpeCDESTR operon mRNAs were upregulated while the cpeBcpeA mRNAs were downregulated in the cpeT mutant strain when compared with WT, suggesting that CpeT may also play a direct or indirect regulatory role in transcription of these operons or their mRNA stability, in addition to its role as a PEB lyase for Cys-165 on ß-PE.

CpeY is a phycoerythrobilin lyase for cysteine 82 of the phycoerythrin I α-subunit in marine Synechococcus.

Marine Synechococcus are widespread in part because they are efficient at harvesting available light using their complex antenna, or phycobilisome, composed of multiple phycobiliproteins and bilin chromophores. Over 40% of Synechococcus strains are predicted to perform a type of chromatic acclimation that alters the ratio of two chromophores, green-light-absorbing phycoerythrobilin and blue-light-absorbing phycourobilin, to optimize light capture by phycoerythrin in the phycobilisome. Lyases are enzymes which catalyze the addition of bilin chromophores to specific cysteine residues on phycobiliproteins and are involved in chromatic acclimation. CpeY, a candidate lyase in the model strain Synechococcus sp. RS9916, added phycoerythrobilin to cysteine 82 of only the α subunit of phycoerythrin I (CpeA) in the presence or absence of the chaperone-like protein CpeZ in a recombinant protein expression system. These studies demonstrated that recombinant CpeY attaches phycoerythrobilin to as much as 72% of CpeA, making it one of the most efficient phycoerythrin lyases characterized to date. Phycobilisomes from a cpeY- mutant showed a near native bilin composition in all light conditions except for a slight replacement of phycoerythrobilin by phycourobilin at CpeA cysteine 82. This demonstrates that CpeY is not involved in any chromatic acclimation-driven chromophore changes and suggests that the chromophore attached at cysteine 82 of CpeA in the cpeY- mutant is ligated by an alternative phycoerythrobilin lyase. Although loss of CpeY does not greatly inhibit native phycobilisome assembly in vivo, the highly active recombinant CpeY can be used to generate large amounts of fluorescent CpeA for biotechnological uses.

Proximity channeling during cyanobacterial phycoerythrobilin synthesis.

Substrate channeling is a widespread mechanism in metabolic pathways to avoid decomposition of unstable intermediates, competing reactions, and to accelerate catalytic turnover. During the biosynthesis of light-harvesting phycobilins in cyanobacteria, two members of the ferredoxin-dependent bilin reductases are involved in the reduction of the open-chain tetrapyrrole biliverdin IXα to the pink pigment phycoerythrobilin. The first reaction is catalyzed by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase and produces the unstable intermediate 15,16-dihydrobiliverdin (DHBV). This intermediate is subsequently converted by phycoerythrobilin:ferredoxin oxidoreductase to the final product phycoerythrobilin. Although substrate channeling has been postulated already a decade ago, detailed experimental evidence was missing. Using a new on-column assay employing immobilized enzyme in combination with UV-Vis and fluorescence spectroscopy revealed that both enzymes transiently interact and that transfer of the intermediate is facilitated by a significantly higher binding affinity of DHBV toward phycoerythrobilin:ferredoxin oxidoreductase. Concluding from the presented data, the intermediate DHBV is transferred via proximity channeling.

Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.

Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte Guillardia theta at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/ß/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.

The roles of the chaperone-like protein CpeZ and the phycoerythrobilin lyase CpeY in phycoerythrin biogenesis.

Phycoerythrin (PE) present in the distal ends of light-harvesting phycobilisome rods in Fremyella diplosiphon (Tolypothrix sp. PCC 7601) contains five phycoerythrobilin (PEB) chromophores attached to six cysteine residues for efficient green light capture for photosynthesis. Chromophore ligation on PE subunits occurs through bilin lyase catalyzed reactions, but the characterization of the roles of all bilin lyases for phycoerythrin is not yet complete. To gain a more complete understanding about the individual functions of CpeZ and CpeY in PE biogenesis in cyanobacteria, we examined PE and phycobilisomes purified from wild type F. diplosiphon, cpeZ and cpeY knockout mutants. We find that the cpeZ and cpeY mutants accumulate less PE than wild type cells. We show that in the cpeZ mutant, chromophorylation of both PE subunits is affected, especially the Cys-80 and Cys-48/Cys-59 sites of CpeB, the beta-subunit of PE. The cpeY mutant showed reduced chromophorylation at Cys-82 of CpeA. We also show that, in vitro, CpeZ stabilizes PE subunits and assists in refolding of CpeB after denaturation. Taken together, we conclude that CpeZ acts as a chaperone-like protein, assisting in the folding/stability of PE subunits, allowing bilin lyases such as CpeY and CpeS to attach PEB to their PE subunit.

Pink bacteria-Production of the pink chromophore phycoerythrobilin with Escherichia coli.

Phycoerythrobilin (PEB) is an open-chain tetrapyrrole derived from heme and plays an important role as light-harvesting pigment in the phycobiliproteins of cyanobacteria and red algae. Furthermore, PEB can also function as an antioxidant with potential use as a natural acid stable food colorant. PEB is not commercially available and large, pure quantities can only be obtained by laborious methanolysis of red algae followed by liquid chromatography. Here we describe an improved method for high yield production and purification of PEB in Escherichia coli via heterologous expression where the two required enzymes heme oxygenase and PEB synthase subsequently convert the substrate heme provided by the host cell. Experiments in shaking flasks resulted in the highest product yield of 680.23 ±â€¯42.75 µg PEB per g cell dry weight, by induction with 0.1 mM IPTG. Scale-up to batch-operated fermentation in a 2 L bioreactor reached product concentrations up to 5.02 mg PEB L-1 by adjustment of aeration, induction time, media composition and supplementation of precursors. A further approach included separation of PEB from developed foam above the culture. This enabled continuous product collection during cultivation and simplified product purification. Produced PEB was validated via UV-vis spectroscopy, high pressure liquid chromatography and mass spectrometry.

Evolution and molecular mechanism of four-electron reducing ferredoxin-dependent bilin reductases from oceanic phages.

Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Thus far, seven FDBR families have been identified, each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate. The newest addition to the family is PcyX, originally identified from metagenomics data derived from phage. Phylogenetically, PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin. PcyX, however, converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. Within this study, we aimed at understanding the evolution of catalytic activities within FDBRs using PcyX as an example. Additional members of the PcyX clade and a remote member of the PcyA family were investigated to gain insights into catalysis. Biochemical data in combination with the PcyX crystal structure revealed that a conserved aspartate-histidine pair is critical for activity. Interestingly, the same residues are part of a catalytic Asp-His-Glu triad in PcyA, including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu failed to convert PcyX to a PcyA. Therefore, the change in regiospecificity is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products. ENZYMES: Suggested EC number for PcyX: 1.3.7.6 DATABASES: The PcyX X-ray structure was deposited in the PDB with the accession code 5OWG.

Ferredoxin-dependent bilin reductases in eukaryotic algae: Ubiquity and diversity.

Linear tetrapyrroles (bilins) are produced from heme by heme oxygenase, usually forming biliverdin IXα (BV). Fungi and bacteria use BV as chromophore for phytochrome photoreceptors. Oxygenic photosynthetic organisms use BV as a substrate for ferredoxin-dependent bilin reductases (FDBRs), enzymes that produce diverse reduced bilins used as light-harvesting pigments in phycobiliproteins and as photoactive photoreceptor chromophores. Bilin biosynthesis is essential for phototrophic growth in Chlamydomonas reinhardtii despite the absence of phytochromes or phycobiliproteins in this organism, raising the possibility that bilins are more generally required for phototrophic growth by algae. We here leverage the recent expansion in available algal transcriptomes, cyanobacterial genomes, and environmental metagenomes to analyze the distribution and diversification of FDBRs. With the possible exception of euglenids, FDBRs are present in all photosynthetic eukaryotic lineages. Phylogenetic analysis demonstrates that algal FDBRs belong to the three previously recognized FDBR lineages. Our studies provide new insights into FDBR evolution and diversification.

Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335.

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.