RESUMO
The radiation of Palearctic green toads (Bufotes) holds great potential to evaluate the role of hybridization in phylogeography at multiple stages along the speciation continuum. With fifteen species representing three ploidy levels, this model system is particularly attractive to examine the causes and consequences of allopolyploidization, a prevalent yet enigmatic pathway towards hybrid speciation. Despite substantial efforts, the evolutionary history of this species complex remains largely blurred by the lack of consistency among the corresponding literature. To get a fresh, comprehensive view on Bufotes phylogeography, here we combined genome-wide multilocus analyses (RAD-seq) with an extensive compilation of mitochondrial, genome size, niche modelling, distribution and phenotypic (bioacoustics, morphometrics, toxin composition) datasets, representing hundreds of populations throughout Eurasia. We provide a fully resolved nuclear phylogeny for Bufotes and highlight exceptional cyto-nuclear discordances characteristic of complete mtDNA replacement (in 20% of species), mitochondrial surfing during post-glacial expansions, and the formation of homoploid hybrid populations. Moreover, we traced the origin of several allopolyploids down to species level, showing that all were exclusively fathered by the West Himalayan B. latastii but mothered by several diploid forms inhabiting Central Asian lowlands, an asymmetry consistent with hypotheses on mate choice and Dobzhansky-Muller incompatibilities. Their intermediate call phenotypes potentially allowed for rapid reproductive isolation, while toxin compositions converged towards the ecologically-closest parent. Across the radiation, we pinpoint a stepwise progression of reproductive isolation through time, with a threshold below which hybridizability is irrespective of divergence (<6My), above which species barely admix and eventually evolve different mating calls (6-10My), or can successfully cross-breed through allopolyploidization (>15My). Finally, we clarified the taxonomy of Bufotes (including genetic analyses of type series) and formally described two new species, B. cypriensis sp. nov. (endemic to Cyprus) and B. perrini sp. nov. (endemic to Central Asia). Embracing the genomic age, our framework marks the advent of a new exciting era for evolutionary research in these iconic amphibians.
Assuntos
Evolução Biológica , Bufonidae/fisiologia , Animais , Bufonidae/classificação , Bufonidae/genética , DNA Mitocondrial/genética , Especiação Genética , Tamanho do Genoma , Genoma Mitocondrial , Genômica , Hibridização Genética , Mitocôndrias/genética , Fenótipo , Filogenia , Filogeografia , Análise de Componente Principal , Fatores de TempoRESUMO
Four heme peroxidase superfamilies arose independently in evolution. Only in the peroxidase-cyclooxygenase superfamily the prosthetic group is posttranslationally modified (PTM). As a consequence these peroxidases can form one, two or three covalent bonds between heme substituents and the protein. This may include ester bonds between heme 1- and 5-methyl groups and glutamate and aspartate residues as well as a sulfonium ion link between the heme 2-vinyl substituent and a methionine. Here the phylogeny and physiological roles of representatives of this superfamily, their occurrence in all kingdoms of life, the relevant sequence motifs for definite identification and the available crystal structures are presented. We demonstrate the autocatalytic posttranslational maturation process and the impact of the covalent links on spectral and redox properties as well as on catalysis, including Compound I formation and reduction by one- and two-electron donors. Finally, we discuss the evolutionary advantage of these PTMs with respect to the proposed physiological functions of the metalloenzymes that range from antimicrobial defence in innate immunity to extracellular matrix formation and hormone biosynthesis.
Assuntos
Biocatálise , Heme/metabolismo , Peroxidases/química , Peroxidases/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Sequência Conservada , HumanosRESUMO
Chinese softshell turtles (Pelodiscus spp.) are widely distributed, ranging from the Amur and Ussuri Rivers in the Russian Far East through the Korean Peninsula, Japan, and eastern, central, and southern China to southern Vietnam. In East and Southeast Asia, Chinese softshell turtles are traditionally exploited for food and have been farm-bred in China since the Spring and Autumn Period, more than 2400 years ago. Currently, the annual production of Pelodiscus amounts to 340,000 t in China alone. Using mitochondrial DNA (2428 bp) and five nuclear loci (3704 bp), we examined broad sampling of wild and farm-bred Pelodiscus to infer genetic and taxonomic differentiation. We discovered four previously unknown mitochondrial lineages, all from China. One lineage from Jiangxi is deeply divergent and sister to the mitochondrial lineage of Pelodiscus axenaria. The nuclear loci supported species status for P. axenaria and the new lineage from Jiangxi. Pelodiscus maackii and P. parviformis, both harboring distinct mitochondrial lineages, were not differentiated from P. sinensis in the studied nuclear markers. The same is true for two new mitochondrial lineages from Zhejiang, China, represented by only one individual each, and another new lineage from Anhui, Guangdong, Jiangxi and Zhejiang, China. However, Vietnamese turtles yielding a mitochondrial lineage clustering within P. sinensis were distinct in nuclear markers, suggesting that these populations could represent another unknown species with introgressed mitochondria. Its species status is also supported by the syntopic occurrence with P. sinensis in northern Vietnam and by morphology. In addition, we confirmed sympatry of P. axenaria and P. parviformis in Guangxi, China, and found evidence for sympatry of P. sinensis and the new putative species from Jiangxi, China. We also discovered evidence for hybridization in turtle farms and for the occurrence of alien lineages in the wild (Zhejiang, China), highlighting the risk of genetic pollution of native stock. In the face of the large-scale breeding of Pelodiscus, we claim that the long-term survival of distinct genetic lineages and species can only be assured when an upscale market segment for pure-bred softshell turtles is established, making the breeding of pure lineages lucrative for turtle farms. Our findings underline that the diversity of Pelodiscus is currently underestimated and threatened by anthropogenic admixture. We recommend mass screening of genetic and morphological variation of Chinese softshell turtles as a first step to understand and preserve their diversity.
Assuntos
Biodiversidade , Cruzamento , Tartarugas/classificação , Tartarugas/genética , Animais , China , DNA Mitocondrial/genética , Repetições de Microssatélites/genética , FilogeniaRESUMO
The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in Escherichia coli, fully reconstituted to the respective heme b proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme b and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Heme/metabolismo , Peroxidase/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Heme/química , Heme/genética , Ligantes , Peroxidase/química , Peroxidase/genéticaRESUMO
The most striking feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO) is the existence of covalent bonds between the prosthetic group and the protein, which has a strong impact on their (electronic) structure and biophysical and chemical properties. Recently, a novel bacterial heme peroxidase with high structural and functional similarities to LPO was described. Being released from Escherichia coli, it contains mainly heme b, which can be autocatalytically modified and covalently bound to the protein by incubation with hydrogen peroxide. In the present study, we investigated the reactivity of these two forms in their ferric, compound I and compound II state in a multi-mixing stopped-flow study. Upon heme modification, the reactions between the ferric proteins with cyanide or H2O2 were accelerated. Moreover, apparent bimolecular rate constants of the reaction of compound I with iodide, thiocyanate, bromide, and tyrosine increased significantly and became similar to LPO. Kinetic data are discussed and compared with known structure-function relationships of the mammalian peroxidases LPO and myeloperoxidase.
Assuntos
Escherichia coli/enzimologia , Heme/química , Peroxidase/química , Peroxidases/química , Brometos/química , Catálise , Cianetos/química , Elétrons , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Iodetos/química , Ferro/química , Lactoperoxidase/química , Modelos Químicos , Oxirredução , Oxigênio/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Espectrofotometria , Tiocianatos/química , Tirosina/químicaRESUMO
Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (-145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.
Assuntos
Proteínas de Bactérias/química , Brometos/química , Cianobactérias/enzimologia , Heme/química , Peroxidase/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Brometos/metabolismo , Cianobactérias/genética , Estabilidade Enzimática/fisiologia , Heme/genética , Heme/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Peroxidase/genética , Peroxidase/metabolismoRESUMO
Despite the relevance of chemical communication in vertebrates, comparative examinations of macroevolutionary trends in chemical signaling systems are scarce. Many turtle and tortoise species are reliant on chemical signals to communicate in aquatic and terrestrial macrohabitats, and many of these species possess specialized integumentary organs, termed mental glands (MGs), involved in the production of chemosignals. We inferred the evolutionary history of MGs and tested the impact of macrohabitat on their evolution. Inference of ancestral states along a time-calibrated phylogeny revealed a single origin in the ancestor of the subclade Testudinoidea. Thus, MGs represent homologous structures in all descending lineages. We also inferred multiple independent losses of MGs in both terrestrial and aquatic clades. Although MGs first appeared in an aquatic turtle (the testudinoid ancestor), macrohabitat seems to have had little effect on MG presence or absence in descendants. Instead, we find clade-specific evolutionary trends, with some clades showing increased gland size and morphological complexity, whereas others exhibiting reduction or MG loss. In sister clades inhabiting similar ecological niches, contrasting patterns (loss vs. maintenance) may occur. We conclude that the multiple losses of MGs in turtle clades have not been influenced by macrohabitat and that other factors have affected MG evolution.
Assuntos
Evolução Biológica , Tegumento Comum/fisiologia , Feromônios/química , Tartarugas/fisiologia , Comunicação Animal , Animais , Fenômenos Biomecânicos , Ecossistema , Tegumento Comum/anatomia & histologia , Feromônios/biossíntese , Filogenia , Transdução de Sinais/genética , Tartarugas/anatomia & histologiaRESUMO
Turtles demonstrate variability in sex determination and, hence, constitute an excellent model for the evolution of sex chromosomes. Notably, the sex determination of the freshwater turtles from the family Chelidae, a species-rich group with wide geographical distribution in the southern hemisphere, is still poorly explored. Here we documented the presence of an XX/XY sex determination system in seven species of the Australasian chelid genera Chelodina, Emydura, and Elseya by conventional (karyogram reconstruction, C-banding) and molecular cytogenetic methods (comparative genome hybridization, in situ hybridization with probes specific for GATA microsatellite motif, the rDNA loci, and the telomeric repeats). The sex chromosomes are microchromosomes in all examined species of the genus Chelodina. In contrast, the sex chromosomes are the 4th largest pair of macrochromosomes in the genera Emydura and Elseya. Their X chromosomes are submetacentric, while their Y chromosomes are metacentric. The chelid Y chromosomes contain a substantial male-specific genomic region with an accumulation of the GATA microsatellite motif, and occasionally, of the rDNA loci and telomeric repeats. Despite morphological differences between sex chromosomes, we conclude that male heterogamety was likely already present in the common ancestor of Chelodina, Emydura and Elseya in the Mesozoic period.
Assuntos
Evolução Molecular , Genoma , Cromossomos Sexuais/genética , Cromossomo X/genética , Cromossomo Y/genética , Animais , Feminino , Cariótipo , Masculino , Repetições de Microssatélites , Processos de Determinação Sexual , TartarugasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Telomeres are nucleoprotein complexes protecting chromosome ends in most eukaryotic organisms. In addition to chromosome ends, telomeric-like motifs can be accumulated in centromeric, pericentromeric and intermediate (i.e., between centromeres and telomeres) positions as so-called interstitial telomeric repeats (ITRs). We mapped the distribution of (TTAGGG)n repeats in the karyotypes of 30 species from nine families of turtles using fluorescence in situ hybridization. All examined species showed the expected terminal topology of telomeric motifs at the edges of chromosomes. We detected ITRs in only five species from three families. Combining our and literature data, we inferred seven independent origins of ITRs among turtles. ITRs occurred in turtles in centromeric positions, often in several chromosomal pairs, in a given species. Their distribution does not correspond directly to interchromosomal rearrangements. Our findings support that centromeres and non-recombining parts of sex chromosomes are very dynamic genomic regions, even in turtles, a group generally thought to be slowly evolving. However, in contrast to squamate reptiles (lizards and snakes), where ITRs were found in more than half of the examined species, and birds, the presence of ITRs is generally rare in turtles, which agrees with the expected low rates of chromosomal rearrangements and rather slow karyotype evolution in this group.
Assuntos
Centrômero/genética , Sequências Repetitivas de Ácido Nucleico/genética , Telômero/genética , Tartarugas/genética , Animais , Feminino , Hibridização in Situ Fluorescente , Cariótipo , Lagartos/genética , Masculino , Cromossomos Sexuais/genética , Serpentes/genéticaRESUMO
For a long time, turtles of the family Geoemydidae have been considered exceptional because representatives of this family were thought to possess a wide variety of sex determination systems. In the present study, we cytogenetically studied Geoemyda spengleri and G. japonica and re-examined the putative presence of sex chromosomes in Pangshura smithii. Karyotypes were examined by assessing the occurrence of constitutive heterochromatin, by comparative genome hybridization and in situ hybridization with repetitive motifs, which are often accumulated on differentiated sex chromosomes in reptiles. We found similar karyotypes, similar distributions of constitutive heterochromatin and a similar topology of tested repetitive motifs for all three species. We did not detect differentiated sex chromosomes in any of the species. For P. smithii, a ZZ/ZW sex determination system, with differentiated sex chromosomes, was described more than 40 years ago, but this finding has never been re-examined and was cited in all reviews of sex determination in reptiles. Here, we show that the identification of sex chromosomes in the original report was based on the erroneous pairing of chromosomes in the karyogram, causing over decades an error cascade regarding the inferences derived from the putative existence of female heterogamety in geoemydid turtles.
RESUMO
Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to the heme and spanning the distal and proximal sides were also investigated. In the wild-type enzyme, the EPR signal of the catalases and peroxidases (typical) Compound I intermediate [Fe(IV)=O por.+] was observed. Two protein-based radical intermediates were also detected and identified as a Tyr. and a Trp. . The site of Trp. is proposed to be Trp 106, a residue belonging to the conserved short stretch in catalase-peroxidases and located at a 7-8 A distance to the heme propionate groups. An extensive hydrogen-bonding network on the heme distal side, involving Trp122, His123, Arg119, seven structural waters, the heme 6-propionate group, and Trp106, is proposed to have a key role on the formation of the tryptophanyl radical. We used high-field EPR spectroscopy (95-285 GHz) to resolve the g-anisotropy of the protein-based radicals in Synechocystis catalase-peroxidase. The broad gx component of the HF EPR spectrum of the Tyr. in Synechocystis catalase-peroxidase was consistent with a distributed electropositive protein environment to the tyrosyl radical.
Assuntos
Proteínas de Bactérias/química , Cianobactérias/enzimologia , Heme/química , Peroxidases/química , Anisotropia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Temperatura Baixa , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Radicais Livres/metabolismo , Heme/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Peroxidases/genética , Peroxidases/metabolismo , Conformação Proteica , Triptofano/químicaRESUMO
Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors. Exchange of tyrosine 249 in Synechocystis KatG, a distal side residue found in all as yet sequenced KatGs, had dramatic consequences on the bifunctional activity and the spectral features of the redox intermediate compound II. The Y249F variant lost catalase activity but retained a peroxidase activity (substrates o-dianisidine, pyrogallol, guaiacol, tyrosine, and ascorbate) similar to the wild-type protein. In contrast to wild-type KatG and similar to monofunctional peroxidases, the formation of the redox intermediate compound I could be followed spectroscopically even by addition of equimolar hydrogen peroxide to ferric Y249F. The corresponding bimolecular rate constant was determined to be (1.1 +/- 0.1) x 107 m-1 s-1 (pH 7 and 15 degrees C), which is typical for most peroxidases. Additionally, for the first time a clear transition of compound I to an oxoferryl-like compound II with peaks at 418, 530, and 558 nm was monitored when one-electron donors were added to compound I. Rate constants of reaction of compound I and compound II with tyrosine ((5.0 +/- 0.3) x 104 m-1 s-1 and (1.7 +/- 0.4) x 102 m-1 s-1) and ascorbate ((1.3 +/- 0.2) x 104 m-1 s-1 and (8.8 +/- 0.1) x 101 m-1 s-1 at pH 7 and 15 degrees C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure.
Assuntos
Proteínas de Bactérias , Cianobactérias/enzimologia , Peroxidases/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Dados de Sequência Molecular , Peroxidases/química , Homologia de Sequência de Aminoácidos , Espectrofotometria UltravioletaRESUMO
Structural and biochemical characterization of aspartate 152 at the distal heme side of catalase-peroxidase (KatG) from Synechocystis PCC 6803 reveals an important functional role for this residue. In the wild-type protein, the side chain carboxyl group of Asp152 is 7.8 A apart from the heme iron and is hydrogen-bonded to two water molecules and a KatG-specific large loop. We have prepared the site-specific variants Asp152Asn, Asp152Ser, Asp152Trp, and Pro151Ala. Exchange of Asp152 exhibited dramatic consequences on the bifunctional activity of this unique peroxidase. The turnover number of catalase activity of Asp152Asn is 2.7%, Asp152Ser 5.7%, and Asp152Trp is 0.6% of wild-type activity. By contrast, the peroxidase activity of the Asp152 variants was 2-7 times higher than that of wild-type KatG or Pro151Ala. The KatG-specific pH profile of the catalase activity was completely different in these variants and exchange of Asp152 made it possible to follow the transition of the ferric enzyme to the redox intermediate compound I by hydrogen peroxide spectroscopically and to determine the corresponding bimolecular rate constant to be 7.5 x 10(6) M(-1) s(-1) (pH 7 and 15 degrees C). The reactivity of compound I toward aromatic one-electron donors was enhanced in the Asp152 variants compared with the wild-type protein, whereas the reactivity toward hydrogen peroxide was dramatically decreased. A mechanism for the hydrogen peroxide oxidation, which is different from monofunctional catalases and involves the distal residues Trp122 and Asp152, is proposed.
Assuntos
Ácido Aspártico/química , Proteínas de Bactérias , Catalase/metabolismo , Cianobactérias/enzimologia , Peroxidases/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/genética , Catálise , Dicroísmo Circular , Escherichia coli/enzimologia , Heme/química , Peróxido de Hidrogênio/metabolismo , Cinética , Modelos Químicos , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Oxirredução , Peroxidases/química , Peroxidases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrofotometria UltravioletaRESUMO
Catalase-peroxidases (KatGs) are unique in exhibiting an overwhelming catalase activity and a peroxidase activity of broad specificity. Similar to other peroxidases the distal histidine in KatGs forms a hydrogen bond with an adjacent conserved asparagine. To investigate the catalytic role(s) of this potential hydrogen bond in the bifunctional activity of KatGs, Asn153 in Synechocystis KatG was replaced with either Ala (Asn153-->Ala) or Asp (Asn153-->Asp). Both variants exhibit an overall peroxidase activity similar with wild-type KatG. Cyanide binding is monophasic, however, the second-order binding rates are reduced to 5.4% (Asn153-->Ala) and 9.5% (Asn153-->Asp) of the value of native KatG [(4.8 +/- 0.4) x 105 m-1.s-1 at pH 7 and 15 degrees C]. The turnover number of catalase activity of Asn153-->Ala is 6% and that of Asn153-->Asp is 16.5% of wild-type activity. Stopped-flow analysis of the reaction of the ferric forms with H2O2 suggest that exchange of Asn did not shift significantly the ratio of rates of H2O2-mediated compound I formation and reduction. Both rates seem to be reduced most probably because (a) the lower basicity of His123 hampers its function as acid-base catalyst and (b) Asn153 is part of an extended KatG-typical H-bond network, the integrity of which seems to be essential to provide optimal conditions for binding and oxidation of the second H2O2 molecule necessary in the catalase reaction.