Molecular cloning and expression analysis of a new bilin lyase: the cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314

To study the assembly of phycocyanin β subunit, the gene cpcT was first cloned from Arthrospira platensis FACHB314. To explore the function of cpcT, the DNA of phycocyanin β subunit and cpcT were transformed into Escherichia coli BL21 with the plasmid pET-hox1-pcyA, which contained the genes hemeoxygenase 1 (Hox1) and ferredoxin oxidoreductase (PcyA) needed to produce phycocyanobilin. The transformed strains showed specific phycocyanin fluorescence, and the fluorescence intensity was stronger than the strains with only phycocyanin β subunit, indicating that CpcT can promote the assembly of phycocyanin to generate fluorescence. To study the possible binding sites of apo-phycocyanin and phycocyanobilin, the Cys-82 and Cys-153 of the β subunit were individually mutated, giving two kinds of mutants. The results show that Cys-153 maybe the active site for β subunit binding to phycocyanobilins, which is catalyzed by CpcT in A. platensis FACHB314.     

Biogenesis of phycobiliproteins: I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phyococyanobilin lyase specific for beta-phycocyanin and allophycocyanin subunits

Phycobilin lyases covalently attach phycobilin chromophores to apo-phycobiliproteins (PBPs). Genome analyses of the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 identified three genes, denoted cpcS-I, cpcU, and cpcV, that were possible candidates to encode phycocyanobilin (PCB) lyases. Single and double mutant strains for cpcS-I and cpcU exhibited slower growth rates, reduced PBP levels, and impaired assembly of phycobilisomes, but a cpcV mutant had no discernable phenotype. A cpcS-I cpcU cpcT triple mutant was nearly devoid of PBP. SDS-PAGE and mass spectrometry demonstrated that the cpcS-I and cpcU mutants produced an altered form of the phycocyanin (PC) beta subunit, which had a mass approximately 588 Da smaller than the wild-type protein. Some free PCB (mass = 588 Da) was tentatively detected in the phycobilisome fraction purified from the mutants. The modified PC from the cpcS-I, cpcU, and cpcS-I cpcU mutant strains was purified, and biochemical analyses showed that Cys-153 of CpcB carried a PCB chromophore but Cys-82 did not. These results show that both CpcS-I and CpcU are required for covalent attachment of PCB to Cys-82 of the PC beta subunit in this cyanobacterium. Suggesting that CpcS-I and CpcU are also required for attachment of PCB to allophycocyanin subunits in vivo, allophycocyanin levels were significantly reduced in all but the CpcV-less strain. These conclusions have been validated by in vitro experiments described in the accompanying report (Saunée, N. A., Williams, S. R., Bryant, D. A., and Schluchter, W. M. (2008) J. Biol. Chem. 283, 7513-7522). We conclude that the maturation of PBP in vivo depends on three PCB lyases: CpcE-CpcF, CpcS-I-CpcU, and CpcT.     

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.     

Lyase activities of CpcS- and CpcT-like proteins from Nostoc PCC7120 and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin beta-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 beta-phycoerythrocyanin (PecB) or beta-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine beta155 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:alpha-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.     

Identification of amino acid residues essential to the activity of lyase CpcT1 from Nostoc sp. PCC7120

The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1.     

R-phycocyanin II, a new phycocyanin occurring in marine Synechococcus species. Identification of the terminal energy acceptor bilin in phycocyanins

A new member of the phycocyanin family of phycobiliproteins, R-phycocyanin II (R-PC II) has been discovered in several strains of marine Synechococcus sp. R-PC II has absorption maxima at 533 and 554 nm, a subsidiary maximum at 615 nm, and a fluorescence emission maximum at 646 nm. It is the first phycoerythrobilin (PEB)-containing phycocyanin of cyanobacterial origin. The purified protein is made up of alpha and beta subunits in equal amounts and is in an (alpha beta)2 aggregation state. The alpha and beta subunits of this protein are homologous to the corresponding subunits of previously described C- and R-phycocyanins as assessed by amino-terminal sequence determination and analyses of sequences about sites of bilin attachment. R-PC II carries phycocyanobilin (PCB) at beta-84 and PEB at alpha-84 and beta-155 (residue numbering is that for C-phycocyanin), whereas in C-phycocyanin PCB is present at all three positions. In R-phycocyanin, the bilin distribution is alpha-84 (PCB), beta-84 (PCB), beta-155 (PEB). In both R-phycocyanin and R-phycocyanin II excitation at 550 nm, absorbed primarily by PEB groups, leads to emission at 625 nm from PCB. These comparative data support the conclusion that the invariant beta-84 PCB serves as the terminal energy acceptor in phycocyanins.     

Molecular characterization of a novel gene from Synechocystis sp. PCC 6803

A novel gene slr2049 was identified in Synechococcus sp. PCC7002 by homologous alignment. The features and possible functions of slr2049 gene were predicted by bioinformatics analysis. The function of slr2049 was analyzed in vitro with a heterologous Escherichia coli system with plasmids conferring biosynthesis of phycocyanobilin (PCB) and of the acceptor proteins, β-phycocyanin (CpcB). The resulting products were evaluated with SDS-PAGE and absorption spectra. The function of slr2049 was further analyzed via site-directed mutations. Two mutants, slr2049 (W14L) and slr2049 (Y132S) were generated. The results showed that Slr2049 could catalyze the chromophorylation of CpcB. Compared to wild type, mutant Slr2049 (W14L) had red-shifted absorbance maxima and was not highly fluorescent as the wild-type. However, mutant Slr2049 (Y132S) was almost the same as the wild-type. In conclusion, our study suggests that we have cloned a novel gene and this gene may play an important role in attachment of the chromophores to the apo-proteins.     

Integration of Apo-α-Phycocyanin into Phycobilisomes and Its Association with FNRL in the Absence of the Phycocyanin α-Subunit Lyase (CpcF) in Synechocystis sp. PCC 6803

Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR_L) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR_L accumulated. FNR_L has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNR_L and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.     

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.     

Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain C-phycocyanin and allophycocyanin in a molar ratio of approximately 3.8:1, a minor biliprotein, allophycocyanin B, and nonpigmented polypeptides of 75, 33, 30, and 27 kilodaltons. A nitrosoguanidine-induced mutant AN112 produces altered phycobilisomes with the molar ratio of C-phycocyanin to allophycocyanin reduced to approximately 1.4:1 and without any of the 33- and 30-kilodalton polypeptides. The mutant and wild type phycobilisomes contain the same molar amount of the 75- and 27-kilodalton polypeptides relative to allophycocyanin. As seen by electron microscopy, the allophycocyanin-containing core of the mutant and of the wild type phycobilisomes appears the same. In some views of the core, each of the two core units in the mutant particles can be seen to consist of four discs approximately 3 nm thick. In wild type phycobilisomes five or six rods, made up of two to six stacked discs (11.5 X 6 nm) are attached to the core. In the mutant, no such rods are seen; rather, single disc-shaped elements, ranging from two to six in number, are found attached. Spectroscopic measurements show that the assembly form of phycocyanin in the mutant phycobilisomes differs from that in the wild type particles but reveal no difference in the organization of the core elements. These results indicate that the portions of the rod substructures of wild type phycobilisomes, beyond the disc proximal to the core, are made up of phycocyanin and the 33- and 30-kilodalton polypeptides. Emission from phycocyanin is a significant component in the fluorescence from isolated Synechococcus 6301 phycobilisomes and indicates an upper limit of 94% for the efficiency of energy transfer from phycocyanin to allophycocyanin and allophycocyanin B in these particles.     

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).     

Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F

To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.     

Spectroscopic properties of tetrapyrroles on denatured biliproteins

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.     

A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant.

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.     

Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cysteine-beta84 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-beta84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-beta84 of the apo-beta-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 degrees C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3 microm, and a kcat = 1.7 x 10(-5) and 1.1 x 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.     

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.     

Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.

The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.     

The crystal structure of a novel unmethylated form of C-phycocyanin,a possible connector between cores and rods in pycobilisomes

A novel fraction of c-phycocyanin from the thermophilic cyanobacterium Thermosynechcoccus vulcanus, with an absorption maxima blue-shifted to 612 nm (PC612), has been purified from allophycocyanin and crystallized. The crystals belong to the P63 space group with cell dimensions of 153 A x 153 A x 59 A with a single (alphabeta) monomer in the asymmetric unit, resulting in a solvent content of 65%, and diffract to 2.7 A. The PC612 crystal structure has been determined by molecular replacement and refined to a crystallographic R-factor of 20.9% (Rfree = 27.8%). The crystal packing in this form shows that the PC612 form of phycocyanin does not associate into hexamers and that its association with adjacent trimers in the unit cell is very different from that found in a previously determined structure of the normal form of T. vulcanus phycocyanin, which absorbs at 620 nm. Analysis of the PC612 structure shows that the alpha subunits, which typically form the interface between two trimers within a hexamer, have a high degree of flexibility, as indicated by elevated B-factors in portions of helices B, E, and G. Examination of calculated electron density omit maps shows that unlike all other structures of phycobiliproteins determined so far, the Asnbeta72 residue is not methylated, explaining the blue-shift in its absorption spectra. On the basis of the results presented here, we suggest that this new form of trimeric phycocyanin may constitute a special minor component of the phycobilisome and may form the contact between the phycocyanin rods and the allophycocyanin core.     

Heterologous assembly and rescue of stranded phycocyanin subunits by expression of a foreign cpcBA operon in Synechocystis sp. strain 6803.

Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membrane-associated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin beta subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin alpha and beta subunits assemble with endogenous phycobilisome components. The phycocyanin alpha subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.