PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents. The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.
The structure of phycoviolobilin, the photoactive chromophore of K K-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a v v3,3 1 -double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-K K-phycoerythrocyanin (PecA). In the absence of the components of K K-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (V V max = 641 nm) and low photoactivity ( 6 10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (V V max = 565 nm) and high photoreversible photochromism (100% type I) has been identified as integral K K-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.z 2000 Federation of European Biochemical Societies.
Transcription of structural genes required for phospholipid biosynthesis in the yeast Saccharomyces cerevisiae is repressed by high concentrations of inositol and choline. The ICRE (inositol/choline‐responsive element), which is necessary and sufficient for regulation by phospholipid precursors, functions as a binding site for the heterodimeric Ino2/Ino4 activator. ICRE‐dependent transcription becomes constitutive in the absence of the Opi1 repressor. Opi1 contains a leucine zipper motif and two glutamine‐rich stretches. In this work we describe a molecular analysis of OPI1 function and expression. Opi1 mutant variants altered at the leucine zipper and a glutamine‐rich region, respectively, were no longer functional repressors. In contrast, an Opi1 deletion variant lacking the N‐terminal 106 amino acids still mediated negative regulation. Although the leucine zipper suggests that Opi1 may act as a DNA‐binding protein, our data do not support a direct interaction with the ICRE. Despite its function as an antagonist of INO2 and INO4, expression of OPI1 is stimulated by an upstream ICRE. Overexpression of OPI1 under control of the GAL1 promoter severely inhibited activation of ICRE‐dependent genes, leading to inositol‐requiring cells. Growth inhibition of GAL1–OPI1 was observed with INO2 and INO4 alleles activated by either the natural promoter or a heterologous control region. Although induction of GAL1–OPI1 strongly repressed ICRE‐dependent gene expression, the concentration of the Ino2/Ino4 activator remained unchanged. This finding suggests that differential expression of phospholipid biosynthetic genes may occur even in the presence of a constant amount of the specific activator. Copyright © 1999 John Wiley & Sons, Ltd.
Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilinphycoerythrocyanin-a84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-a-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg 2+ or Mn 2+ , were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (K m ¼ 12-16 lM, depending on [PecA], k cat 1.2 · 10 )4 AEs )1 ) and the apoprotein (K m ¼ 2.4 lM at 14 lM PCB, k cat ¼ 0.8 · 10 )4 AEs )1 ). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEClyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.
Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 alpha-subunit, and the C-13(1) keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the beta-subunit. Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13(1) keto carbonyl and a methyl hydrogen of the alanine replacing serine(-4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13(1) not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 A in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups.
In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane serine residue at position ؊4 relative to the liganding histidine of the ␣-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the residue at this position in the helix. A hydrogen bond between the hydroxy group of the serine and the 13 1 keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the serine residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.
Chlorophyll is attached to apoprotein in diastereotopically distinct ways, by -and ␣-ligation. Both the -and ␣-ligated chlorophylls of photosystem I are shown to have ample contacts to apoprotein within their proteinaceous binding sites, in particular, at C-13 of the isocyclic ring. The H-bonding patterns for the C-13 1 oxo groups, however, are clearly distinct for the -ligated and ␣-ligated chlorophylls. The -ligated chlorophylls frequently employ their C-13 1 oxo in H-bonds to neighboring helices and subunits. In contrast, the C-13 1 oxo of ␣-ligated chlorophylls are significantly less involved in H-bonding interactions, particularly to neighboring helices. Remarkably, in the peripheral antenna, light harvesting complex (LH2) from Rhodobacter sphaeroides, a single mutation in the ␣-subunit, introduced to eliminate H-bonding to the -bacteriochlorophyll-B850, which is ligated in the "-position," results in significant thermal destabilization of the LH2 in the membrane. In addition, in comparison with wild type LH2, the expression level of the LH2 lacking this H-bond is significantly reduced. These findings show that H-bonding to the C-13 1 keto group of -ligated (bacterio)-chlorophyll is a key structural motif and significantly contributes to the stability of bacteriochlorophyll proteins in the native membrane. Our analysis of photosystem I and II suggests that this hitherto unrecognized motif involving H-bonding to -ligated chlorophylls may be equally critical for the stable assembly of the inner core antenna of these multicomponent chlorophyll proteins.
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