Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins.
ABSTRACT: The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arabidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant-specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arabidopsis, while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper-binding amino acids. The recombinant copper binding domain of Arabidopsis uclacyanin 1 was expressed, purified, and shown to bind a copper atom in a fashion known as "blue" or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin 1 rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild-type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron-transfer proteins participating in long-range electron-transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.
Project description:BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section. CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences.
Project description:The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.
Project description:BACKGROUND: Phytocyanins (PCs) are plant-specific blue copper proteins involved in electron transport, and a large number of known PCs are considered to be chimeric arabinogalactan proteins (AGPs). To date there has not been a genome-wide overview of the OsPC gene family. Therefore, as the first step and a useful strategy to elucidate the functions of OsPCs, there is an urgent need for a thorough genome-wide analysis of this gene family. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a total of 62 OsPC genes were identified through a comprehensive bioinformatics analysis of the rice (Oryza sativa L.) genome. Based on phylogeny and motif constitution, the family of OsPCs was classified into three subclasses: uclacyanin-like proteins (OsUCLs), stellacyanin-like proteins (OsSCLs) and early nodulin-like proteins (OsENODLs). Structure and glycosylation prediction indicated that 46 OsPCs were glycosylphosphatigylinositol-anchored proteins and 38 OsPCs were chimeric AGPs. Gene duplication analysis revealed that chromosomal segment and tandem duplications contributed almost equally to the expansion of this gene family, and duplication events were mostly happened in the OsUCL subfamily. The expression profiles of OsPC genes were analyzed at different stages of vegetative and reproductive development and under abiotic stresses. It revealed that a large number of OsPC genes were abundantly expressed in the various stages of development. Moreover, 17 genes were regulated under the treatments of abiotic stresses. CONCLUSIONS/SIGNIFICANCE: The genome-wide identification and expression analysis of OsPC genes should facilitate research in this gene family and give new insights toward elucidating their functions in higher plants.
Project description:Interactions of the axial ligand with its blue copper center are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of the blue copper center with a weak axial ligand to a green copper center containing a medium strength axial ligand has been demonstrated in cupredoxins, converting the blue copper center to a red copper center with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (R(L)) of absorption at 447 nm over that at 621 nm. Whereas no axial Cu-S(Cys121) interaction in Met121Cys was detectable by extended X-ray absorption fine structure (EXAFS) spectroscopy at pH 5, similar to what was observed in native azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 A is clearly visible at higher pH. Despite the higher R(L) and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A( parallel) = 54 x 10(-4) cm(-1) at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A(parallel) = 87 x 10(-4) cm(-1) at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with a nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (R(L)= 1.5, and A(parallel) = 180 x 10(-4) cm(-1)) and Cu-S(Cys) distance (2.22 A) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys or homocysteine resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 +/- 3 mV for Met121Cys azurin and 113 +/- 6 mV for Met121Hcy azurin at pH 7. The results strongly support the "coupled distortion" model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.
Project description:Phytocyanins (PCs) are plant-specific blue copper proteins, which play essential roles in electron transport. While the origin and expansion of this gene family is not well-investigated in plants. Here, we investigated their evolution by undertaking a genome-wide identification and comparison in 10 plants: Arabidopsis, rice, poplar, tomato, soybean, grape, maize, Selaginella moellendorffii, Physcomitrella patens, and Chlamydomonas reinhardtii. We found an expansion process of this gene family in evolution. Except PCs in Arabidopsis and rice, which have described in previous researches, a structural analysis of PCs in other eight plants indicated that 292 PCs contained N-terminal secretion signals and 217 PCs were expected to have glycosylphosphatidylinositol-anchor signals. Moreover, 281 PCs had putative arabinogalactan glycomodules and might be AGPs. Chromosomal distribution and duplication patterns indicated that tandem and segmental duplication played dominant roles for the expansion of PC genes. In addition, gene organization and motif compositions are highly conserved in each clade. Furthermore, expression profiles of maize PC genes revealed diversity in various stages of development. Moreover, all nine detected maize PC genes (ZmUC10, ZmUC16, ZmUC19, ZmSC2, ZmUC21, ZmENODL10, ZmUC22, ZmENODL13, and ZmENODL15) were down-regulated under salt treatment, and five PCs (ZmUC19, ZmSC2, ZmENODL10, ZmUC22, and ZmENODL13) were down-regulated under drought treatment. ZmUC16 was strongly expressed after drought treatment. This study will provide a basis for future understanding the characterization of this family.
Project description:BACKGROUND:Pollen tube formation and growth are crucial steps that lead to seed production. Despite the importance of pollen tube growth, the molecular mechanisms implicated in its spatial and temporal control are not fully known. In this study, we found an uclacyanin gene, OsUCL8, that regulates pollen intine deposition and pollen tube growth. FINDINGS:The overexpression of OsUCL8 led to a striking irregularity in pollen tube growth and pollination and thus affected the seed setting rate in rice; many pollen tubes appeared to lose the ability to grow directly into the style. Conversely, plants with OsUCL8 knocked out and plants overexpressing miR408, a negative regulator of OsUCL8, had vigorous pollens with a higher germination rate. We further demonstrated that OsUCL8 mainly affects pollen intine formation. The addition of Vitamin B1 (VB1) significantly contributed to the germination of OXUCL8 pollen grains, suggesting that OsUCL8 could be associated with VB1 production. Using a yeast two-hybrid system, we revealed that OsUCL8 interacts with the protein OsPKIWI, a homolog of the Arabidopsis FNRL protein. We thus hypothesized that OsUCL8 might regulate the production of VB components by interacting with OsPKIWI. This study revealed a novel molecular mechanism of pollen tube growth regulation. CONCLUSIONS:The rice plantacyanin family member OsUCL8 plays an important role in pollen tube formation and growth and, in turn, regulates fertility and the seed setting rate.
Project description:miR408 is highly conserved among different plant species and targets transcripts encoding copper-binding proteins. The function of miR408 in reproductive development remains largely unclear despite it being known to play important roles during vegetative development in Arabidopsis. Here, we show that transgenic Arabidopsis plants overexpressing MIR408 have altered morphology including significantly increased leaf area, petiole length, plant height, flower size, and silique length, resulting in enhanced biomass and seed yield. The increase in plant size was primarily due to cell expansion rather than cell proliferation, and was consistent with higher levels of myosin gene expression and gibberellic acid (GA) measured in transgenic plants. In addition, photosynthetic rate was significantly increased in the MIR408-overexpressing plants, as manifested by higher levels of chloroplastic copper content and plastocyanin (PC) expression. In contrast, overexpression of miR408-regulated targets, Plantacyanin and Laccase 13, resulted in reduced biomass production and seed yield. RNA-sequencing revealed that genes involved in primary metabolism and stress response were preferentially enriched in the genes upregulated in MIR408-overexpressing plants. These results indicate that miR408 plays an important role in regulating biomass and seed yield and that MIR408 may be a potential candidate gene involved in the domestication of agricultural crops.
Project description:A combination of spectroscopies and density functional theory calculations indicate that there are large temperature-dependent absorption spectral changes present in green nitrite reductases (NiRs) due to a thermodynamic equilibrium between a green and a blue type 1 (T1) copper site. The axial methionine (Met) ligand is unconstrained in the oxidized NiRs, which results in an enthalpically favored (DeltaH approximately 4.6 kcal/mol) Met-bound green copper site at low temperatures, and an entropically favored (TDeltaS approximately 4.5 kcal/mol, at room temperature) Met-elongated blue copper site at elevated temperatures. In contrast to the NiRs, the classic blue copper sites in plastocyanin and azurin show no temperature-dependent behavior, indicating that a single species is present at all temperatures. For these blue copper proteins, the polypeptide matrix opposes the gain in entropy that would be associated with the loss of the weak axial Met ligand at physiological temperatures by constraining its coordination to copper. The potential energy surfaces of Met binding indicate that it stabilizes the oxidized state more than the reduced state. This provides a mechanism to tune down the reduction potential of blue copper sites by >200 mV.
Project description:The Cu-SCys interaction is known to play a dominant role in defining the type 1 (T1) blue copper center with respect to both its electronic structure and electron transfer function. Despite this importance, its role has yet to be probed by mutagenesis studies without dramatic change of its T1 copper character. We herein report replacement of the conserved Cys112 in azurin with the nonproteinogenic amino acid homocysteine. Based on electronic absorption, electron paramagnetic resonance, and extended x-ray absorption fine structural spectroscopic studies, this variant displays typical type 1 copper site features. Surprisingly, instead of increasing the strength of the Cu-sulfur interaction by the introduction of the extra methylene group, the Cys112Hcy azurin showed a decrease in the covalent interaction between SHcy and Cu(II) when compared with the WT SCys-Cu(II) interaction. This is likely due to geometric adjustment of the center that resulted in the copper ion moving out of the trigonal plane defined by two histidines and one Hcy and closer to Met121. These structural changes resulted in an increase of reduction potential by 35 mV, consistent with lower Cu-S covalency. These results suggest that the Cu-SCys interaction is close to being optimal in native blue copper protein. It also demonstrates the power of using nonproteinogenic amino acids in addressing important issues in bioinorganic chemistry.
Project description:Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.