Isolation and biliprotein characterization of phycobilisomes from the thermophilic cyanobacterium Mastigocladus laminosus Cohn

A method for the effective isolation of functionally intact phycobilisomes from the thermophilic cyanobacterium M. laminosus is presented, using an unconventional high buffer molarity for stabilizing the aggregates and introducing a DNAse treatment of the disrupted cells to obtain sharp banding of the phycobilisomes in the linear sucrose density gradients.The structural integrity of the isolated phycobilisomes is demonstrated by a fluorescence emission maximum at 673 nm of aggregated allophycocyanin and by electron microscopy.Besides C-phycocyanin and allophycocyanin, phycoerythrocyanin is a constituent pigment of the phycobilisomes. These pigments indicated in the absorption spectrum of phycobilisomes with a maximum at 610 nm and two shoulders at 650 and 580 nm, respectively, were characterized by spectral data and isoelectric points.     

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.     

Recent advances in production,purification and applications of phycobiliproteins

An obligatory sunlight requirement for photosynthesis has exposed cyanobacteria to different quantity and quality of light. Cyanobacteria can exhibit efficient photosynthesis over broad region(450 to 650 nm) of solar spectrum with the help of brilliantly coloured pigment proteins called phycobiliproteins(PBPs). Besides light-harvesting, PBPs are found to involve in several life sustaining phenomena including photoprotection in cyanobacteria. The unique spectral features(like strong absorbance and fluorescence), proteineous nature and, some imperative properties like hepato-protective, antioxidants, anti-inflammatory and anti-aging activity of PBPs enable their use in food, cosmetics, pharmaceutical and biomedical industries. PBPs have been also noted to show beneficial effect in therapeutics of some disease like Alzheimer and cancer. Such large range of applications increases the demand of PBPs in commodity market. Therefore, the large-scale and coast effective production of PBPs is the real need of time. To fulfil this need, many researchers have been working to find the potential producer of PBPs for the production and purification of PBPs. Results of these efforts have caused the inventions of some novel techniques like mixotrophic and heterotrophic strategies for production and aqueous two phase separation for purification purpose. Overall, the present review summarises the recent findings and identifies gaps in the field of production, purification and applications of this biological and economically important proteins.     

Bacterial versus human sphingosine-1-phosphate lyase (S1PL) in the design of potential S1PL inhibitors

A series of potential active-site sphingosine-1-phosphate lyase (S1PL) inhibitors have been designed from scaffolds 1 and 2, arising from virtual screening using the X-ray structures of the bacterial (StS1PL) and the human (hS1PL) enzymes. Both enzymes are very similar at the active site, as confirmed by the similar experimental kinetic constants shown by the fluorogenic substrate RBM13 in both cases. However, the docking scoring functions used probably overestimated the weight of electrostatic interactions between the ligands and key active-site residues in the protein environment, which may account for the modest activity found for the designed inhibitors. In addition, the possibility that the inhibitors do not reach the enzyme active site should not be overlooked. Finally, since both enzymes show remarkable structural differences at the access channel and in the proximity to the active site cavity, caution should be taken when designing inhibitors acting around that area, as evidenced by the much lower activity found in StS1PL for the potent hS1PL inhibitor D.     

Dynamic aspects of phycobilisome structure

In exponentially growing cells of Synechococcus sp. 6301, over 95% of the phycobiliproteins are located in phycobilisomes, and the remainder is present in the form of low molecular weight aggregates. In addition to the subunits of the phycobiliproteins (C-phycocyanin, allophycocyanin, allophycocyanin B), the phycobilisomes of this unicellular cyanobacterium contain five non-pigmented polypeptides. During the initial phase of starvation (24 h after removal of combined nitrogen from the growth medium), the phycobiliproteins in the low molecular weight fraction largely disappeared. Phycocyanin was lost more rapidly from this fraction than allophycocyanin. Simultaneous changes in the phycobilisome were (1) a decrease in sedimentation coefficient, (2) a decrease in phycocyanin: allophycocyanin ratio, (3) a shift in the fluorescence emission maximum from 673 to 676 nm, and (4) a selective complete loss of a 30,000 dalton non-pigmented polypeptide. Upon extensive nitrogen starvation (72 h), the intracellular level of phycocyanin decreased by over 30-fold. These results indicate that in the early stage of nitrogen starvation, the free phycobiliproteins of the cell are degraded, as well as a significant proportion of the phycocyanin from the periphery of the phycobilisome. However, the structures partially depleted of phycocyanin still function efficiently in energy transfer. On extended starvation, total degradation of residual phycobilisomes takes place, possibly in conjunction with the detachment of these structures from the thylakoids.None of the effects of the absence of combined nitrogen were seen when cells were starved in the presence of chloramphenicol, or in a methionine auxotroph starved for methionine.Abbreviations Used NaK-PO₄ NaH₂PO₄ titrated with K₂HPO₄ to a given pH - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)aminomethane     

Isolation and characterization of phycoerythrocyanin and chromatic adaptation of the thermophilic cyanobacterium Mastigocladus laminosus

The thermophilic cyanobacterium Mastigocladus laminosus exhibits chromatic adaptation: In green light the production of the phycobiliprotein phycoerythrocyanin is enhanced drastically.Phycoerythrocyanin was characterised with respect to its molecular weight, isoelectric point, absorption spectra and size of its aggregates. The two subunits of the protein were separated and characterised according to these criteria. Their chromophore contents, amino acid compositions and N-terminal amino acid sequences were also determined. The sequences were compared with those of allophycocyanin and C-phycocyanin from Mastigocladus laminosus.Abbreviations PEC phycoerythrocyanin - -PEC -subunit of PEC - -PEC -subunit of PEC - PE phycoerythrin - PC phycocyanin - APC allophycocyanin - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - chl a chlorophyll a     

Correlation between pectate lyase activity and ability of diazotrophic Klebsiella oxytoca VN 13 to penetrate into plant tissues

Diazotrophic Klebsiella oxytoca VN 13 was able to lyse pectate, but the lytic activity of cultures grown on non-selective media was weak, and the enzyme was located mainly inside the cells. A small fraction of the population (10^-6 to 10^-5) was able to grow in a selective medium with sodium polygalacturonate as sole carbon source, and produced increased amounts of the pectinolytic enzyme pectate lyase. When wheat seeds were inoculated with these bacteria, increased levels of this enzyme correlated with a higher rate of internal colonization of wheat roots and with stimulation of wheat growth resulting in higher biomass. This suggests that colonization occurs via lyzed pectin layers. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structure and Mechanism of the Phycobiliprotein Lyase CpcT

Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965–983).     

Extraction and purification of phycocyanin from Calothrix sp.

The extraction and purification of phycocyanin from Calothrix sp., cyanobacteria isolated from rice fields in Cuernavaca, Morelos, Mexico is described. Phycocyanin was extracted with 2 mg of lysozyme/g wet biomass, and purified by anion chromatography using Q-Sepharose fast-flow (Pharmacia^®, 1.5 cm×10 cm) column and hydrophobic interaction chromatography with methyl macro-prep (Bio-Rad^®, 1.5 cm×20 cm) column. The purified protein showed a pI of 5.2 and has two subunits with apparent molecular mass of 21–17 kDa each. The estimated molecular mass of native purified phycocyanin was 114 kDa, suggesting a stereochemistry of (β)₃.     

Biliprotein assembly in the hemidiscoidal phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus Cohn. Characterization of dissociation products with special reference to the peripheral phycoerythrocyanin-phycocyanin complexes

The dissociation products of isolated phycobilisomes of Mastigocladus laminosus were separated and analyzed by ultracentrifugation and, in part, by isoelectric focusing. With the exception of the allophycocyanin core, the sedimentation constants of peripheral phycocyanin- and phycoerythrocyanin-phycocyanin complexes lay in the range of 6 to 17S. The latter was represented by a 17S aggregate of two hexameric phycocyanins (dodecamer, dipartite unit). A complex with an absorption maximum at 610 nm (phycocyanin) and a shoulder at 580 nm (phycoerythrocyanin), a fluorescence emission maximum at 645 nm and a sedimentation constant of 11 S is described as a heterogeneously composed hexamer of ()₃-phycoerythrocyanin-()₃-phycocyanin. It was stable under extended dissociation in the cold and under isoelectric focusing. An aggregate of 14 S with an absorption maximum at 576 nm and a shoulder in the fluorescence emission spectrum at 625 nm (phycoerythrocyanin) in addition to the maximum at 645 nm (phycocyanin) is interpreted as a polar phycoerythrocyanin/ phycoerythrocyanin-phycocyanin complex. Combining these complexes with phycocyanin dodecamers creates peripheral rods of the phycobilisome. A proposal of the phycobiliprotein distribution within the phycobilisome of M. laminosus is presented.Abbreviations APC allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin     

Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.     

Isolation and characterization of heterocysts from Anabaena sp. strain CA

A comparative study has been made on the pigment composition and nitrogenase activity of whole filaments and isolated beterocysts from a mutant strain of Anabaena CA. The whole cell absorption spectra of intact filaments and isolated heterocysts showed close resemblance especially between 550–700 nm region. On a quantitative basis the chlorophyll a content was found almost equal between the vegetative cell and heterocyst but the c-phycocyanin content in the heterocyst was about 1/2 that of the vegetative cell. The purification of the phycobiliprotein on DEAE-cellulose showed the presence of c-phycocyanin (_max 615 nm) and allophycocyanin (_max 645 nm, shoulder 620 nm). Isolated heterocysts under H₂ showed acetylene reduction rates of 57 nmol C₂H₄/mg dry wt·min (342 mol C₂H₄/mg chl a·h), whereas intact filaments reduced at the rate of 18 nmol C₂H₄/mg dry wt·min (108 mol C₂H₄/mg chl a·h). This rate accounts for 30% recovery of nitrogenase activity in isolated heterocysts compared to whole filaments. The activity was strictly light dependent and was linear under H₂ for more than 3 h. Addition of as little as 5% H₂ under argon stimulated the C₂H₂ reductionseveral fold. The acetylene reduction (nitrogenase activity) also showed tolerance to 5% added O₂ either under H₂ or argon. The results suggest that the heterocyst of Anabaena CA-V is different in some characteristics (viz., higher endogenous C₂H₂ reduction rate, prolonged activity and higher levels of phycobiliproteins) than those reported in other Anabaena species.     

Allophycocyanin trimer stability and functionality are primarily due to polar enhanced hydrophobicity of the phycocyanobilin binding pocket

Allophycocyanin (APC) is the primary pigment-protein component of the cores of the phycobilisome antenna complex. In addition to an extremely high degree of amino acid sequence conservation, the overall structures of APC from both mesophilic and thermophilic species are almost identical at all levels of assembly, yet APC from thermophilic organisms should have structural attributes that prevent thermally induced denaturation. We determined the structure of APC from the thermophilic cyanobacterium Thermosynechococcus vulcanus to 2.9 Å, reaffirming the conservation of structural similarity with APC from mesophiles. We provide spectroscopic evidence that T. vulcanus APC is indeed more stable at elevated temperatures in vitro, when compared with the APC from mesophilic species. APC thermal and chemical stability levels are further enhanced when monitored in the presence of high concentrations of buffered phosphate, which increases the strength of hydrophobic interactions, and may mimic the effect of cytosolic crowding. Absorption spectroscopy, size-exclusion HPLC, and native gel electrophoresis also show that the thermally or chemically induced changes in the APC absorption spectra that result in the loss of the prominent 652-nm band in trimeric APC are not a result of physical monomerization. We propose that the bathochromic shift that occurs in APC upon trimerization is due to the coupling of the hydrophobicity of the α84 phycocyanobilin cofactor environment created by a deep cleft formed by the β subunit with highly charged flanking regions. This arrangement also provides the additional stability required by thermophiles at elevated temperatures. The chemical environment that induces the bathochromic shift in APC trimers is different from the source of shifts in the absorption of monomers of the terminal energy acceptors APC^B and L_CM, as visualized by the building of molecular models.     

Transient state characteristics of adaptation to changes in light conditions for the cyanobacterium Oscillatoria agardhii

Transitions in growth irradiance level from 92 to 7 Em^-2 s^-1 and vice versa caused changes in the pigment contents and photosynthesis of Oscillatoria agardhii. The changes in chlorophyll a and C-phycocyanin contents during the transition from high to low irradiance (HL) were reflected in photosynthetic parameters. In the LH transition light utilization efficiencies of the cells changed faster than pigment contents. This appeared to be related to the lowering of light utilization efficiencies of photosynthesis. As a possible explanation it was hypothesized that excess photosynthate production led to feed back inhibition of photosynthesis. Time-scales of changes in the maximal rate of O₂ evolution were discussed as changes in the number of reaction centers of photosystem II in relation to photosynthetic electron transport. Parameters that were subject to change during irradiance transitions obeyed first order kinetics, but hysteresis occurred when comparing HL with LH transients. Interpretation of first order kinetic analysis was discussed in terms of adaptive response vs changes in growth rate.Non-standard abbreviations Chla chlorophyll a - CPC C-phycocyanin - PS II photosystem II - PS I photosystem I - RC II reaction center of photosystem II - P photosynthetic O₂-evolution - I irradiance, Em^-2 s^-1 - light utilization efficiency of cells, mmol O₂·mg dry wt^-1·h^-1/Em^-2 s^-1 - light utilization efficiency of photosynthetic apparatus, mol O₂·mol Chla ^-1·h^-1/Em^-2 s^-1 - P_max maximal rate of O₂ evolution by cells, mol O₂·mg dry wt^-1·h^-1 - P_max maximal rate of O₂ evolution by photosynthetic apparatus, mol O₂·mol·Chla ^-1·h^-1 - LL low light, E m^-2 s^-1 - HL high light, E m^-2 s^-1 - LH low to high light transition - HL high to low light transition - k specific rate of adaptation, h^-1 - specific growth rate, h^-1 - Q pool size of cell constituent, mol·mg dry wt^-1 - q net synthesis rate of cell constituent, mol·mg dry wt^-1·h^-1     

Native and in vitro-associated phycobilisomes of Nostoc sp. Composition,energy transfer,and effect of antibodies

The relationship of the structure and function of the light-harvesting antennae in the blue-green alga Nostoc sp. was further elucidated by reconstitution experiments. Separated phycoerythrin-phycocyanin complexes and allophycocyanin fractions were reassociated as described earlier (Canaani, O., Lipschultz, C.A. and Gantt, E. (1980) FEBS Lett. 115, 225–229) into functional phycobilisomes with a 70% yield. Native and reassociated physobilisomes had molar ratios of about 1.4:1.1:1.0 of phycoerythrin:phycocyanin:allophycocyanim. Energy transfer was demonstrated by their fluorescence emission maximum at approx. 675 nm (20°C), and their excitation spectra (emission wavelength 680 nm) which reflected the contribution of the three constitutive phycobiliproteins. Scans of Coomassie blue-stained SDS-polyacrylamide gels showed that the polypeptide composition of native and reassociated phycobilisomes was virtually indistinguishable. Reassociation of phycobilisomes was dependent on the interaction of allophycocyanin and phycocyanin, because it could be blocked with antisera to phycocyanin and allophycocyanin, but not to phycoerythrin. In addition, reassociation did not occur when a 31 000 Da polypeptide, which is part of the phycoerythrin-phycocyanin complex, was reduced in size (by 4000 Da). These results suggest that at least two domains are required for functional reassociation of phycobilisomes involving phycocyanin and allophycocyanin.     

Characterization and implications of Ca2+ binding to pectate lyase C

Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.     

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.     

Elucidation of the mode of substrate binding to hydroxynitrile lyase from Hevea brasiliensis

The hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) is used as a catalyst in enantiospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we modeled Hb-HNL substrate complexes that have as yet proven inaccessible to experimental structure determination and were able to identify two binding modes for the natural substrate acetone cyanohydrin in docking simulations. Discrimination of the two alternatives was achieved by modeling complexes with two different chiral cyanohydrins followed by an analysis of the respective relative binding energies from molecular mechanics and thermodynamic integration. Only for one of the alternative binding modes the experimentally established S-selectivity of the enzyme was correctly predicted. Our results yielded further support for an enzymatic mechanism involving the catalytic triad Ser80, His235, and Asp207 as a general acid/base. A pivotal role was ascribed to Lys236, which seems to be crucial for enzymatic activity at low pH values. In addition, the modeling calculations provided possible explanations for the observed substrate and enantioselectivity of the enzyme that rationalize available mutational data and will be the basis for future protein engineering efforts.     

A stable mercury-containing complex of the organomercurial lyase MerB: catalysis, product release, and direct transfer to MerA

Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism.