3,356 research outputs found
bilin' full
No full textbileRegatta Day] if a man chooses to become "bilin' full, ye know," at nine o'clock in the morning and dead to the world at ten, it's his own affair, . . .PRINTED ITEMW. J. KIRWIN NOV 12 1964Not usedNot usedNot use
Syntheses of biliverdins (Bilin-1,19-diones) from a, c-biladienes and b-bilenes
No full textTreatment of readily accessible 1,19-bis(t-butoxycarbonyl)-a, c-biladienes or b-bilenes with bromine in trifluoroacetic acid affords excellent yields of biliverdins (bilin-1,19-diones). © The Royal Society of Chemistry, 1982
The D-ring, Not the A-ring, Rotates in Synechococcus OS-B' Phytochrome
No full textPhytochrome photoreceptors in plants and microorganisms switch photochromically between two states, controlling numerous important biological processes. Although this phototransformation is generally considered to involve rotation of ring D of the tetrapyrrole chromophore, Ulijasz et al. (Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera, M., Markley, J. L., and Vierstra, R. D. (2010) Nature 463, 250–254) proposed that the A-ring rotates instead. Here, we apply magic angle spinning NMR to the two parent states following studies of the 23-kDa GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain fragment of phytochrome from Synechococcus OS-B′. Major changes occur at the A-ring covalent linkage to the protein as well as at the protein residue contact of ring D. Conserved contacts associated with the A-ring nitrogen rule out an A-ring photoflip, whereas loss of contact of the D-ring nitrogen to the protein implies movement of ring D. Although none of the methine bridges showed a chemical shift change comparable with those characteristic of the D-ring photoflip in canonical phytochromes, denaturation experiments showed conclusively that the same occurs in Synechococcus OS-B′ phytochrome upon photoconversion. The results are consistent with the D-ring being strongly tilted in both states and the C15=C16 double bond undergoing a Z/E isomerization upon light absorption. More subtle changes are associated with the A-ring linkage to the protein. Our findings thus disprove A-ring rotation and are discussed in relation to the position of the D-ring, photoisomerization, and photochromicity in the phytochrome family
Phycobiliprotein Lyases
No full textPhycobilins are light harvesting pigments of cyanobacteria and red algae. In cyanobacteria, four phycobiliproteins are organized in phycobilisomes: phycocyanin (PC), allophycocyanin (APC), and often also phycoerythrocyanin (PEC) or phycoerythrin (PE). Their phycobilin chromophores, linear tetrapyrroles, are generally bound to the apoprotein at conserved positions by cysteinyl thioether linkages. A final step in phycobiliprotein biosynthesis is the post-translational phycobilin addition to the various biliproteins. In vivo, the correct attachment of most chromophores is catalyzed by binding-site and chromophore-specific lyases. Only two such lyases, which both belong to the E/F-type were known at the beginning of this work. Two additional types, S/(U)-type and T-type lyase, have been characterized during this work. In addition, the correct structures of the products from all three lyase types have been verified, and evidence was obtained for the reaction mechanisms.
This characterization relied on two methodological advances. The first is the use of a multi-plasmidic expression system for reconstitution of phycobiliproteins in E. coli. After cloning of apophycobiliprotein genes, phycobilin biosynthesis genes and (putative) lyase genes from several cyanobacteria, various phycobiliproteins could be biosynthesized in the heterologous E. coli system using dual plasmids containing the respective genes. This heterologous system produces higher yields than the in vitro reconstitution, it is nearly devoid of spontaneous binding, better reproducible, and more easily controlled. The second methodological advance is the consequent use of a combination of chromatographic, electrophoretic and spectroscopic tools that allowed a full characterization of the structure and binding sites of attached chromophores. This included, besides optical spectroscopy, in particular mass and magnetic resonance (1H-NMR) spectroscopy.
Using the unmodified genes coding for both subunits of PEC, as well as their cystein mutants, three lyases were identified for the three binding site. Besides the already known isomerizing lyase, PecE/PecF, for Cys-84 of α-PEC, these are the two new lyases, CpcT (all5339) for Cys-153 of β-PEC, and CpcS (alr0617) for Cys-82 of β-PEC. The spectroscopic analysis proved that the chromophores (PCB and PVB)are correctly attached to these three binding sites.
Similarly, three lyases were identified for the three binding sites of CPC. The well known heterodimeric lyase (CpcE/CpcF) catalyzes the covalent attachment of PCB to αC84 of CPC, CpcS catalyses the site-selective attachment of PCB to cysteine-β84 in CpcB; and CpcT for cysteine-β155 of CpcB. CpcE/F is specific for CpcA, while CpcS and CpcT can react with both CpcB and PecB. We also tested the lyase activity of the deoxyhyposyl-hydroxylase (DOHH) from the malaria parasite, Plasmodium falciparum. This enzyme has Heat-like repeats that are characteristic for the E/F-type lyases, but it had not chromophore-attaching activity.
The substrate specificity of the new lyase, CpcS (coded by alr0617), was further tested with APC subunits; It is very unspecific with regard to the acceptor protein and attaches PCB to ApcA1, ApcB, ApcD ApcF, as well as to the product of an additional gene, apcA2; of unknown function that is highly homologous to apcA1 coding for the APC α-subunit. Obviously, this lyase has a much broader substrate specificity than the E/F-type lyases, but it has high site-specificity, attaching the chromophore exclusively to the Cys-84 (consensus sequence) binding site of the APC subunits.
CpcS from Anabaena PCC7120 is a relatively simple system, it acts as a monomer, and does not require any cofactors. CpcS binds PCB rapidly (<1s) and relatively strongly, but probably non-covalently. The chromophore is bound in an extended conformation similar to that in phycobiliproteins, but only poorly fluorescent. The extended conformation is supported by binding studies with a conformationally locked chromophore, 15Za-PCB, which also binds rapidly and non-covalently to CpcS and gives a product with similar spectral properties as PCB-CpcS. Upon addition of apo-biliproteins to the PCB-CpcS (or 15Za-PCB-CpcS) complex, the chromophore is transferred to the latter much more slowly (~1 hr), indicating that chromophorylated CpcS is an intermediate in the enzymatic reaction.
There are distinct differences in the absorption, extinction coefficient in acidic methanol and pKa of the free 15Za-PCB, compared with that of free PCB, which are probably due to a shift in the pK-values by about 1 pH unit.
Nucleophilic addition products of PCB were characterized that are formed spontaneously or by the lyases, and gave first indications for a mechanistic model for the lyases. The first nucleophile was imidazole, which is a model for histidine. Two imidazole-PCB adducts were prepared and the structures determined by MS and NMR spectroscopy. Surprisingly, the chromophore is isomerized in this reaction to a 2,22 H –bilin termed iso-phycocyanobilin (iPCB). CpcS not only can promote covalent binding of PCB to imidazole, but also catalyses the transfer of the chromophore of the formed iPCB-imidazole to the cysteine84 of acceptor apoprotein, CpcB. During this transfer reaction, the chromophore is re-isomerized to PCB, to yield CpcB-C84-PCB. It indicates that chromophorylation by CpcS might then involve a histidine-bound intermediate; this could be a model for the reaction catalyzed by CpcS.
The second nucleophile was mercaptoethanol, as a model for cysteine. In the ME and PCB reaction system, two isomers each of isomeric PVB-ME and iPCB-ME were obtained in a non-enzymatic reaction. The chromophore of the two complexes can be transferred to cysteine-84 of CpcB, yielding CpcB-C84-PCB and CpcB-C84-PVB. In the presence of the lyase, CpcS, only the iPCB adducts are formed. It indicates autocatalytical chromophorylation might then involve a thiol-chromophore intermediate; this could be a model for the chromophorylation reaction. At the same, we propose a possible generalized catalytic mechanism for the non-isomerizing heterodimeric lyase, CpcE/CpcF, and its isomerizing homolog, PecE/PecF
Identification of the Deprotonated Pyrrole Nitrogen of the Bilin-Based Photoreceptor by Raman Spectroscopy with an Advanced Computational Analysis
No full textPhytochrome
and cyanobacteriochrome utilize a linear methine-bridged
tetrapyrrole (bilin) to control numerous biological processes. They
show a reversible photoconversion between two spectrally distinct
states. This photocycle is initiated by a CC double-bond photoisomerization
of the bilin followed by its thermal relaxations with transient and/or
stationary changes in the protonation state of the pyrrole moiety.
However, it has never been identified which of the four pyrrole nitrogen
atoms is deprotonated. Here, we report a resonance Raman spectroscopic
study on cyanobacteriochrome RcaE, which has been proposed to contain
a deprotonated bilin for its green-absorbing 15Z state.
The observed Raman spectra were well reproduced by a simulated structure
whose bilin B ring is deprotonated, with the aid of molecular dynamics
and quantum mechanics/molecular mechanics calculations. The results
revealed that the deprotonation of B and C rings has the distinct
effect on the overall bilin structure, which will be relevant to the
color tuning and photoconversion mechanisms of the phytochrome superfamily.
Furthermore, this study documents the ability of vibrational spectroscopy
combined with the advanced spectral analysis to visualize a proton
of a cofactor molecule embedded in a protein moiety
Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus
No full textCyanobacterial phycobiliproteins
have evolved to capture light
energy over most of the visible spectrum due to their bilin chromophores,
which are linear tetrapyrroles that have been covalently attached
by enzymes called bilin lyases. We report here the crystal structure
of a bilin lyase of the CpcS family from Thermosynechococcus
elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and
belongs to the lipocalin structural family. TeCpcS-III
catalyzes both cognate as well as noncognate bilin attachment to a
variety of phycobiliprotein subunits. TeCpcS-III
ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to
the alpha and beta subunits of allophycocyanin and to the beta subunit
of phycocyanin at the Cys82-equivalent position in all cases. The
active form of TeCpcS-III is a dimer, which is consistent
with the structure observed in the crystal. With the use of the UnaG
protein and its association with bilirubin as a guide, a model for
the association between the native substrate, phycocyanobilin, and TeCpcS was produced
Green, red, near-infrared: biophysical investigations on bacterial bilin-binding photoreceptors
No full textBacterial photoreceptors binding open-chain tetrapyrroles (bilins) as chromophores are related to plant phytochromes (phy) as they are photochromic and the primary photochemistry consists of a Z/E isomerisation around the bilin 15=16 double bond. The chromophore is embedded in all cases within a so-called GAF domain with a typical α/β fold. Different to the canonical plant phys that invariably bind phytochromobilin and switch between a red and a far red absorbing form (R/FR), the bacterial bilin-photoreceptors exhibit a much wider variety of spectroscopic and functional properties, and bind diverse bilin chromophores, e.g. phycocyanobilin (PCB) and biliverdin (BV). In particular, isolated GAF domains of cyanobacteriochromes (CBCRs) take on great importance, because they are photochromic as standalone units. Beyond their intrinsic interest as light-sensing systems in prokaryots, these proteins show features, as a larger fluorescence quantum yield (F) and broader spectral ranges, that render them good candidates for biotechnological applications. Here we report steady-state and time-resolved spectroscopic measurements on selected bacterial bilin-photoreceptors: a. BV-binding phy from Pseudomonas strains with FR/NIR photochromism; b. PCB-binding GAF3 domain of the R/G (Red/Green) switching CBCR 1393 (Slr1393g3) from Synechocystis; c. GAF1 (R/FR) and GAF3 (R/Orange) domains of Anabaena 2699 (All2699). For CBCR GAF domains, both wild-type and mutated proteins, for which the dynamics of light-triggered reactions is altered, were analyzed. Within this pool of bacterial bilin-photoreceptors, a good correlation was found between F and fluorescence lifetimes. Nanosecond time-resolved absorption spectroscopy revealed the kinetics and spectral features of transient species after photoactivation. The most unusual behavior was found for the G-form of Slr1393g3: this form exhibits steady-state fluorescence heterogeneity and an up to now undescribed optical transient with a lifetime of ca. 60 ns at 20°C, upon green-light excitation. This transient was first uncovered by means of a photothermal method, but was now tracked by careful inspections of transient optical kinetic traces. Definitely, further investigations will be necessary to enlighten a possible link between the detected heterogeneity of Slr1393g3-G and this novel transient species
CpeF is the bilin lyase that ligates the doubly linked phycoerythrobilin on -phycoerythrin in the cyanobacterium Fremyella diplosiphon
No full textPhycoerythrin (PE) is a green light–absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, we found that cpeF is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes
CpeF is the Bilin Lyase that Ligates the Doubly Linked Phycoerythrobilin on Phycoerythrin in the Cyanobacterium Fremyella Diplosiphon
No full textPhycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, we found that cpeF is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes
CpeF is the bilin lyase that ligates the doubly linked phycoerythrobilin on -phycoerythrin in the cyanobacterium Fremyella diplosiphon
No full textPhycoerythrin (PE) is a green light–absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, we found that cpeF is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes
- …
