Aminoadipate reductase gene: a new fungal-specific gene for comparative evolutionary analyses

BACKGROUND: In fungi, aminoadipate reductase converts 2-aminoadipate to 2-aminoadipate 6-semialdehyde. However, other organisms have no homologue to the aminoadipate reductase gene and this pathway appears to be restricted to fungi. In this study, we designed degenerate primers for polymerase chain reaction (PCR) amplification of a large fragment of the aminoadipate reductase gene for divergent fungi. RESULTS: Using these primers, we amplified DNA fragments from the archiascomycetous yeast Saitoella complicata and the black-koji mold Aspergillus awamori. Based on an alignment of the deduced amino acid sequences, we constructed phylogenetic trees. These trees are consistent with current ascomycete systematics and demonstrate the potential utility of the aminoadipete reductase gene for phylogenetic analyses of fungi. CONCLUSIONS: We believe that the comparison of aminoadipate reductase among species will be useful for molecular ecological and evolutionary studies of fungi, because this enzyme-encoding gene is a fungal-specific gene and generally appears to be single copy

The primordial metabolism: an ancestral interconnection between leucine, arginine, and lysine biosynthesis

<p>Abstract</p> <p>Background</p> <p>It is generally assumed that primordial cells had small genomes with simple genes coding for enzymes able to react with a wide range of chemically related substrates, interconnecting different metabolic routes. New genes coding for enzymes with a narrowed substrate specificity arose by paralogous duplication(s) of ancestral ones and evolutionary divergence. In this way new metabolic pathways were built up by primordial cells. Useful hints to disclose the origin and evolution of ancestral metabolic routes and their interconnections can be obtained by comparing sequences of enzymes involved in the same or different metabolic routes. From this viewpoint, the lysine, arginine, and leucine biosynthetic routes represent very interesting study-models. Some of the <it>lys</it>, <it>arg </it>and <it>leu </it>genes are paralogs; this led to the suggestion that their ancestor genes might interconnect the three pathways. The aim of this work was to trace the evolutionary pathway leading to the appearance of the extant biosynthetic routes and to try to disclose the interrelationships existing between them and other pathways in the early stages of cellular evolution.</p> <p>Results</p> <p>The comparative analysis of the genes involved in the biosynthesis of lysine, leucine, and arginine, their phylogenetic distribution and analysis revealed that the extant metabolic "grids" and their interrelationships might be the outcome of a cascade of duplication of ancestral genes that, according to the patchwork hypothesis, coded for unspecific enzymes able to react with a wide range of substrates. These genes belonged to a single common pathway in which the three biosynthetic routes were highly interconnected between them and also to methionine, threonine, and cell wall biosynthesis. A possible evolutionary model leading to the extant metabolic scenarios was also depicted.</p> <p>Conclusion</p> <p>The whole body of data obtained in this work suggests that primordial cells synthesized leucine, lysine, and arginine through a single common metabolic pathway, whose genes underwent a set of duplication events, most of which can have predated the appearance of the last common universal ancestor of the three cell domains (Archaea, Bacteria, and Eucaryotes). The model proposes a relative timing for the appearance of the three routes and also suggests a possible evolutionary pathway for the assembly of bacterial cell-wall.</p

Evolution of Lysine Biosynthesis in the Phylum Deinococcus-Thermus

Thermus thermophilus biosynthesizes lysine through the α-aminoadipate (AAA) pathway: this observation was the first discovery of lysine biosynthesis through the AAA pathway in archaea and bacteria. Genes homologous to the T. thermophilus lysine biosynthetic genes are widely distributed in bacteria of the Deinococcus-Thermus phylum. Our phylogenetic analyses strongly suggest that a common ancestor of the Deinococcus-Thermus phylum had the ancestral genes for bacterial lysine biosynthesis through the AAA pathway. In addition, our findings suggest that the ancestor lacked genes for lysine biosynthesis through the diaminopimelate (DAP) pathway. Interestingly, Deinococcus proteolyticus does not have the genes for lysine biosynthesis through the AAA pathway but does have the genes for lysine biosynthesis through the DAP pathway. Phylogenetic analyses of D. proteolyticus lysine biosynthetic genes showed that the key gene cluster for the DAP pathway was transferred horizontally from a phylogenetically distant organism

The hidden universal distribution of amino acid biosynthetic networks: a genomic perspective on their origins and evolution

A core of widely distributed network branches biosynthesizing at least 16 out of the 20 standard amino acids is predicted using comparative genomics

Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

<p><b>Abstract</b></p> <p>Background</p> <p>Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (<it>NPS</it>) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.</p> <p>Results</p> <p>Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to <it>NPS </it>genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.</p> <p>Conclusions</p> <p>This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the α-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.</p

Rapid Pathway Evolution Facilitated by Horizontal Gene Transfers across Prokaryotic Lineages

The evolutionary history of biological pathways is of general interest, especially in this post-genomic era, because it may provide clues for understanding how complex systems encoded on genomes have been organized. To explain how pathways can evolve de novo, some noteworthy models have been proposed. However, direct reconstruction of pathway evolutionary history both on a genomic scale and at the depth of the tree of life has suffered from artificial effects in estimating the gene content of ancestral species. Recently, we developed an algorithm that effectively reconstructs gene-content evolution without these artificial effects, and we applied it to this problem. The carefully reconstructed history, which was based on the metabolic pathways of 160 prokaryotic species, confirmed that pathways have grown beyond the random acquisition of individual genes. Pathway acquisition took place quickly, probably eliminating the difficulty in holding genes during the course of the pathway evolution. This rapid evolution was due to massive horizontal gene transfers as gene groups, some of which were possibly operon transfers, which would convey existing pathways but not be able to generate novel pathways. To this end, we analyzed how these pathways originally appeared and found that the original acquisition of pathways occurred more contemporaneously than expected across different phylogenetic clades. As a possible model to explain this observation, we propose that novel pathway evolution may be facilitated by bidirectional horizontal gene transfers in prokaryotic communities. Such a model would complement existing pathway evolution models

Amino acid transport in Penicillium chrysogenum in relation to precursor supply for beta-lactam production

Penicilline wordt in de industrie gewonnen uit de schimmel Penicillium chrysogenum. In de cellen van deze schimmel vormt het antibioticum zich in enkele stappen. De eerste daarvan is het aaneenkoppelen van drie aminozuurmoleculen, waaronder alfa-aminoadipaat. Die aminozuren worden alledrie door de cellen zelf geproduceerd en hoeven voor de penicillineproductie in principe dus niet 'gevoed' te worden. Maar als men wel alfa-aminoadipaat toevoegt aan het groeimedium, gaat de penicillineproductie omhoog. Dit betekent in elk geval dat alfa-aminoadipaat wordt opgenomen door de cellen. Kern van het promotieonderzoek van Hein Trip is de vraag: hoe wordt het van buiten naar binnen getransporteerd? Celmembranen bevatten verschillende eiwitten die aminozuren kunnen transporteren. In het onderzoek karakteriseerde Trip vier van dit soort eiwitten uit Penicillium chrysogenu. Twee daarvan bleken in staat om alfa-aminoadipaat te transporteren over een celmembraan.

Bioinformatics of genome evolution: from ancestral to modern metabolism

Bioinformatics, that is the interdisciplinary field that blends computer science and biostatistics with biological and biomedical sciences, is expected to gain a central role in next feature. Indeed, it has now affected several fields of biology, providing crucial hints for the understanding of biological systems and also allowing a more accurate design of wet lab experiments. In this work, the analysis of sequence data has be used in different fields, such as evolution (e.g. the assembly and evolution of metabolism), infections control (e.g. the horizontal flow of antibiotic resistance), ecology (bacterial bioremediation)

Aminoadipato-semialdeído sintase (AASS) como um novo alvo terapêutico para epilepsia dependente de piridoxina

Orientador: Paulo Arruda, José Andrés YunesTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A via da sacaropina é considerada a principal rota para o catabolismo de lisina em mamíferos, na qual a enzima Aminoadipato Semialdeído Sintase (AASS) catalisa a oxidação da lisina ao aminoadipato semialdeído (AASA). Este é imediatamente oxidado a ácido aminoadípico (AAA) por ação da enzima Aminoadipato Semialdeído Desidrogenase (AASADH, codificada pelo gene aldh7a1). Em humanos, mutações no gene AASS são a causa da hiperlisinemia, um erro inato do metabolismo, porém benigno. Por outro lado, mutações no gene Aldh7a1 causam Epilepsia Dependente de Piridoxina (PDE), na qual o acúmulo de AASA e sua forma cíclica, Piperideine-6 Carboxilato (P6C), são consideradas as principais causas desta doença. Estes compostos causam depleção de Piridoxal-5¿ fosfato (PLP) do organismo por meio da formação de produtos de condensação com o composto P6C. Apesar de existir uma rota alternativa de degradação de lisina (conhecida como via do pipecolato), hipotetizamos que os níveis elevados de AASA/P6C observados no plasma e urina de pacientes com PDE decorrem, principalmente, da atividade da via da sacaropina, especificamente da enzima AASS. Durante o presente trabalho de doutorado, desenvolvemos um novo método quantitativo de espectrometria de massas (LC-MS/MS) que permitiu a analise dos metabólitos da degradação de lisina em plasma e tecidos de camundongos. Como resultado da quantificação de metabólitos e também experimentos de rastreio de 15N (usando-se injeção de lisina marcada no nitrogênio ? ou ?) sugerimos a enzima AASS como a principal enzima de degradação de lisina do organismo. Desta forma, esta enzima seria a responsável pela síntese da maior parte do AASA/P6C acumulado em pacientes de PDE e, sendo que esta enzima é principalmente hepática e renal, sugerimos estes órgãos como os principais sítios de produção destes compostos tóxicos. Adicionalmente, a ideia de que a via do pipecolato possui apenas um papel minoritário no metabolismo geral de lisina é também suportada por esses experimentos, onde cerca da metade deste composto circulante provavelmente se origina da via da sacaropina. Este efeito também foi observado e discutido por nós em outros modelos biológicos como bactéria (Ruegeria pomeroyi) e plantas (Zea mays). Sugerimos, portanto, que a escolha da AASS como alvo terapêutico para desenvolvimento de inibidores seja uma nova alternativa para tratamento de PDE, pois seria observada redução dos níveis tóxicos de AASA/P6C. Observamos que o uso de shRNA para realizar knockdown da expressão do gene aass em células HEK293T não resultou em nenhum fenótipo detrimental e também não causou indução dos genes da via do pipecolato. Ao estudarmos células primárias de cinco distintos pacientes de PDE (modelo de fibroblastos de pele) observamos que estas células possuem maior sensibilidade à presença de Lisina ao meio de cultura se comparadas à células normais. Como prova de conceito observamos que o knockdown da expressão de aass usando shRNA restaura a sobrevivência das células PDE na presença de altos níveis de lisina. Isso pode sugerir o uso de ASO (Anti-sense Oligonucleotides) ou inibidores da atividade da AASS para tratamento da PDE. Para promover melhor entendimento da bioquímica e biologia estrutural deste novo alvo terapêutico, expressamos, purificamos e obtivemos com sucesso a estrutura cristalográfica do domínio Sacaropina Desidrogenase (SDH) da enzima AASS humana. Propriedades cinéticas e bioquímicas da enzima foram estudadas, bem como foram realizados ensaios de screening de ligantes por desnaturação térmica e ensaios de atividade. Foram feitas várias rodadas de desenho computacional de inibidores baseados na estrutura do sítio ativo com o intuito de usar esta tecnologia para desenhar compostos que atuem competindo com a sacaropina pelo sítio ativoAbstract: The saccharopine pathway is considered the main route for lysine catabolism in higher eukaryotes. In this pathway, the enzyme aminoadipate-semialdehyde synthase (AASS) catalyzes the oxidation of lysine into aminoadipic semialdehyde (AASA). The latter is immediately oxidized to aminoadipic acid (AAA) by aminoadipic semialdehyde dehydrogenase (AASADH). In humans, mutations affecting AASS activity lead to hyperlysinemia, a benign inborn error of metabolism. In contrast, mutations affecting AASADH activity cause pyridoxine dependent epilepsy (PDE), in which the accumulation of AASA and its cyclic form piperideine-6-carboxylate (P6C) are considered the main pathogenic drivers of this disease as it depletes pyridoxal 5?-phosphate (PLP) by formation of Knoevenagel condensation products. Although there exist another lysine catabolic route known as the pipecolate pathway, we hypothesize that the elevated plasma and urine levels of AASA/P6C observed in PDE patients arises predominantly from the saccharopine pathway. In the work comprising this thesis, a series of experiments were carried out employing diverse bilological models to study lysine catabolism to AAA. In addition, a new quantitative LC-MS/MS method was developed, which allowed the analysis of lysine catabolism products in mouse plasma and tissues. The quantification of lysine metabolites and the tracking of N15-labelled lysine suggest that AASS is the main enzyme of lysine degradation. We suggest that the primary source of pathogenic accumulation of AASA/P6C is the liver and the kidney, since they present high levels of AASS activity. In addition, we demonstrate that the pipecolate pathway plays only a minor role in the overall figure of lysine oxidation to AAA. The idea of the predominant role of the sacharopine pathway in lysine degradation was reinforced by studies performed in bacteria and plants and also from the observation that a significant proportion of the circulating pipecolate is actually produced from the saccharopine pathway. Taken together these results may support the hypothesis that targeting AASS inhibition or AASS down-regulation at transcription level would reduce the toxic levels of AASA/P6C and this may result in a better outcome for PDE. We then knocked-down AASS in HEK293T cells and observed that this does not imply in any detrimental phenotype. Then we used primary skin fibroblasts from PDE patients and observed that these cells display reduced viability in the presence of high lysine levels when compared to skin fibroblasts derived from normal children. As proof-of-concept, we recovered the normal cell survival in the presence of high lysine levels by knocking-down AASS expression in skin fibroblasts obtained from PDE patients, which suggest that inhibition of AASS prevents accumulation of AASA/P6C and thus improves cell performance. This may potentially be a new method to treat PDE. In order to understand the biochemistry and structural biology of human AASS as a tool for the design of an enzyme inhibitor, we expressed, purified and obtained the crystallographic structure by X-ray diffraction at 2.6 Å of the saccharopine dehydrogenase (SDH) domain of human AASS. The solved structure consists on a homodimer bound to the cofactor NAD+. Structure-based drug design approaches are being used to design potential ligands that may act as competitive inhibitors to saccharopine binding siteDoutoradoBioinformaticaDoutora em Genética e Biologia Molecular2012/00235-5FAPES