RESUMEN
The polyamine spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homospermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homospermidine, suggesting it replaces spermidine for growth. To test this concept, a budding yeast spermidine auxotrophic strain was found to grow almost normally with homospermidine instead of spermidine. Biosynthesis of spermidine analogue aminopropylcadaverine, but not exogenously provided norspermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the spermidine biosynthetic pathway but retain deoxyhypusine synthase.
Asunto(s)
Eucariontes/metabolismo , Paramecium/genética , Paramecium/metabolismo , Poliaminas/metabolismo , Espermidina/biosíntesis , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Adenosilmetionina Descarboxilasa/química , Adenosilmetionina Descarboxilasa/genética , Adenosilmetionina Descarboxilasa/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Secuencia de Aminoácidos , Animales , Vías Biosintéticas/genética , Cadaverina/análogos & derivados , Cadaverina/biosíntesis , Eucariontes/genética , Fusión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Procesamiento Proteico-Postraduccional , Schistosoma/genética , Alineación de Secuencia , Espermidina/análogos & derivados , Espermidina/farmacología , Espermidina Sintasa/genética , Espermidina Sintasa/metabolismo , Levaduras/efectos de los fármacos , Levaduras/genética , Levaduras/crecimiento & desarrolloRESUMEN
Structural backbones of iron-scavenging siderophore molecules include polyamines 1,3-diaminopropane and 1,5-diaminopentane (cadaverine). For the cadaverine-based desferroxiamine E siderophore in Streptomyces coelicolor, the corresponding biosynthetic gene cluster contains an ORF encoded by desA that was suspected of producing the cadaverine (decarboxylated lysine) backbone. However, desA encodes an l-2,4-diaminobutyrate decarboxylase (DABA DC) homologue and not any known form of lysine decarboxylase (LDC). The only known function of DABA DC is, together with l-2,4-aminobutyrate aminotransferase (DABA AT), to synthesize 1,3-diaminopropane. We show here that S. coelicolor desA encodes a novel LDC and we hypothesized that DABA DC homologues present in siderophore biosynthetic clusters in the absence of DABA AT ORFs would be novel LDCs. We confirmed this by correctly predicting the LDC activity of a DABA DC homologue from a Yersinia pestis siderophore biosynthetic pathway. The corollary was confirmed for a DABA DC homologue, adjacent to a DABA AT ORF in a siderophore pathway in the cyanobacterium Anabaena variabilis, which was shown to be a bona fide DABA DC. These findings enable prediction of whether a siderophore pathway will utilize 1,3-diaminopropane or cadaverine, and suggest that the majority of bacteria use DABA AT and DABA DC for siderophore, rather than norspermidine/polyamine biosynthesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Evolución Molecular , Sideróforos/biosíntesis , Streptomyces coelicolor/enzimología , Anabaena variabilis/química , Anabaena variabilis/enzimología , Anabaena variabilis/genética , Bacterias/química , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Carboxiliasas/genética , Datos de Secuencia Molecular , Filogenia , Poliaminas/metabolismo , Streptomyces coelicolor/genéticaRESUMEN
We have identified gene fusions of polyamine biosynthetic enzymes S-adenosylmethionine decarboxylase (AdoMetDC, speD) and aminopropyltransferase (speE) orthologues in diverse bacterial phyla. Both domains are functionally active and we demonstrate the novel de novo synthesis of the triamine spermidine from the diamine putrescine by fusion enzymes from ß-proteobacterium Delftia acidovorans and δ-proteobacterium Syntrophus aciditrophicus, in a ΔspeDE gene deletion strain of Salmonella enterica sv. Typhimurium. Fusion proteins from marine α-proteobacterium Candidatus Pelagibacter ubique, actinobacterium Nocardia farcinica, chlorobi species Chloroherpeton thalassium, and ß-proteobacterium D. acidovorans each produce a different profile of non-native polyamines including sym-norspermidine when expressed in Escherichia coli. The different aminopropyltransferase activities together with phylogenetic analysis confirm independent evolutionary origins for some fusions. Comparative genomic analysis strongly indicates that gene fusions arose by merger of adjacent open reading frames. Independent fusion events, and horizontal and vertical gene transfer contributed to the scattered phyletic distribution of the gene fusions. Surprisingly, expression of fusion genes in E. coli and S. Typhimurium revealed novel latent spermidine catabolic activity producing non-native 1,3-diaminopropane in these species. We have also identified fusions of polyamine biosynthetic enzymes agmatine deiminase and N-carbamoylputrescine amidohydrolase in archaea, and of S-adenosylmethionine decarboxylase and ornithine decarboxylase in the single-celled green alga Micromonas.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Vías Biosintéticas/genética , Evolución Molecular , Fusión Génica , Putrescina/metabolismo , Espermidina Sintasa/genética , Espermidina/metabolismo , Adenosilmetionina Descarboxilasa/metabolismo , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Espermidina Sintasa/metabolismoRESUMEN
Polyamine biosynthesis in plants differs from other eukaryotes because of the contribution of genes from the cyanobacterial ancestor of the chloroplast. Plants possess an additional biosynthetic route for putrescine formation from arginine, consisting of the enzymes arginine decarboxylase, agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase, derived from the cyanobacterial ancestor. They also synthesize an unusual tetraamine, thermospermine, that has important developmental roles and which is evolutionarily more ancient than spermine in plants and algae. Single-celled green algae have lost the arginine route and are dependent, like other eukaryotes, on putrescine biosynthesis from the ornithine. Some plants like Arabidopsis thaliana and the moss Physcomitrella patens have lost ornithine decarboxylase and are thus dependent on the arginine route. With its dependence on the arginine route, and the pivotal role of thermospermine in growth and development, Arabidopsis represents the most specifically plant mode of polyamine biosynthesis amongst eukaryotes. A number of plants and algae are also able to synthesize unusual polyamines such as norspermidine, norspermine and longer polyamines, and biosynthesis of these amines likely depends on novel aminopropyltransferases similar to thermospermine synthase, with relaxed substrate specificity. Plants have a rich repertoire of polyamine-based secondary metabolites, including alkaloids and hydroxycinnamic amides, and a number of polyamine-acylating enzymes have been recently characterised. With the genetic tools available for Arabidopsis and other model plants and algae, and the increasing capabilities of comparative genomics, the biological roles of polyamines can now be addressed across the plant evolutionary lineage.
Asunto(s)
Evolución Biológica , Chlorophyta/metabolismo , Enzimas/metabolismo , Plantas/metabolismo , Poliaminas/metabolismo , Arabidopsis/metabolismo , Vías Biosintéticas , Bryopsida/metabolismoRESUMEN
Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the alpha-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N(1)-aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the alpha-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some alpha-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Evolución Biológica , Vías Biosintéticas , Poliaminas/metabolismo , Espermidina/análogos & derivados , Bacterias , Cromatografía Líquida de Alta Presión , Modelos Moleculares , Filogenia , Espermidina/metabolismoRESUMEN
Polygalacturonases (PGs) have been proposed to play an important role in the process of cell separation. The Arabidopsis thaliana genome contains 69 annotated genes that by amino acid homology and transcript organization could be classified as putative PGs and these can be grouped into multiple clades. An analysis of five members located in two separate clades, using reporter fusion constructs and reverse transcription-PCR, revealed that whilst these PGs exhibit high sequence similarity they have distinct patterns of spatial and temporal expression. Sites of expression include the aleurone and endosperm cells surrounding the emerging radicle in a germinating seed, the cortical cells adjacent to the developing lateral root, the abscission zones of floral organs, the dehiscence zone of anthers and siliques, and pollen grains. Silencing of an abscission-related PG (At2g41850), using a T-DNA insertion strategy, delayed the time-course of floral organ loss but did not prevent shedding from taking place. These observations are discussed with regard to the contribution that PGs may play during the life cycle of a plant.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Regulación de la Expresión Génica de las Plantas/fisiología , Poligalacturonasa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Separación Celular , Familia de Multigenes , Filogenia , Poligalacturonasa/genéticaRESUMEN
A fundamental issue in the safety assessment of genetically modified crops is the question of whether unintentional changes have occurred in the crop plant as a consequence of the genetic modification. This question was addressed recently by using a powerful metabolite fingerprinting and metabolite profiling method to assess whether genetically modified potatoes are substantially similar to their corresponding conventional cultivars.
Asunto(s)
Plantas Modificadas Genéticamente/metabolismo , Solanum tuberosum/genética , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Ingeniería Genética , Análisis Multivariante , Plantas Modificadas Genéticamente/genética , Análisis de Componente Principal , Solanum tuberosum/metabolismoRESUMEN
A novel form of translational regulation is described for the key polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC). Plant AdoMetDC mRNA 5' leaders contain two highly conserved overlapping upstream open reading frames (uORFs): the 5' tiny and 3' small uORFs. We demonstrate that the small uORF-encoded peptide is responsible for constitutively repressing downstream translation of the AdoMetDC proenzyme ORF in the absence of increased polyamine levels. This first example of a sequence-dependent uORF to be described in plants is also functional in Saccharomyces cerevisiae. The tiny uORF is required for normal polyamine-responsive AdoMetDC mRNA translation, and we propose that this is achieved by control of ribosomal recognition of the occluded small uORF, either by ribosomal leaky scanning or by programmed -1 frameshifting. In vitro expression demonstrated that both the tiny and the small uORFs are translated. This tiny/small uORF configuration is highly conserved from moss to Arabidopsis thaliana, and a more diverged tiny/small uORF arrangement is found in the AdoMetDC mRNA 5' leader of the single-celled green alga Chlamydomonas reinhardtii, indicating an ancient origin for the uORFs.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Poliaminas Biogénicas/metabolismo , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Secuencia de Bases , Poliaminas Biogénicas/farmacología , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Homeostasis , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Cell separation is a critical process that takes place throughout the life cycle of a plant. It enables roots to emerge from germinating seeds, cotyledons, and leaves to expand, anthers to dehisce, fruit to ripen, and organs to be shed. The focus of this review is to examine how processes such as abscission and dehiscence are regulated and the ways new research strategies are helping us to understand the mechanisms involved in bringing about a reduction in cell-to-cell adhesion. The opportunities for using this information to manipulate cell separation for the benefit of agriculture and horticulture are evaluated.