Mannosylglycerate: structural analysis of biosynthesis and evolutionary history

Halophilic and halotolerant microorganisms adapted to thrive in hot environments accumulate compatible solutes that usually have a negative charge either associated with a carboxylic group or a phosphodiester unit. Mannosylglycerate (MG) has been detected in several members of (hyper)thermophilic bacteria and archaea, in which it responds primarily to osmotic stress. The outstanding ability of MG to stabilize protein structure in vitro as well as in vivo has been convincingly demonstrated. These findings led to an increasingly supported link between MG and microbial adaptation to high temperature. However, the accumulation of MG in many red algae has been known for a long time, and the peculiar distribution of MG in such distant lineages was intriguing. Knowledge on the biosynthetic machinery together with the rapid expansion of genome databases allowed for structural and phylogenetic analyses and provided insight into the distribution of MG. The two pathways for MG synthesis have distinct evolutionary histories and physiological roles: in red algae MG is synthesised exclusively via the single-step pathway and most probably is unrelated with stress protection. In contrast, the two-step pathway is strongly associated with osmoadaptation in (hyper)thermophilic prokaryotes. The phylogenetic analysis of the two-step pathway also reveals a second cluster composed of fungi and mesophilic bacteria, but MG has not been demonstrated in members of this cluster; we propose that the synthase is part of a more complex pathway directed at the synthesis of yet unknown molecules containing the mannosyl-glyceryl unit.     

Applied evolutionary theories for engineering of secondary metabolic pathways

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Benzoxazinoid biosynthesis,a model for evolution of secondary metabolic pathways in plants

Benzoxazinoids are secondary metabolites that are effective in defence and allelopathy. They are synthesised in two subfamilies of the Poaceae and sporadically found in single species of the dicots. The biosynthesis is fully elucidated in maize; here the genes encoding the enzymes of the pathway are in physical proximity. This “biosynthetic cluster” might facilitate coordinated gene regulation. Data from Zea mays, Triticum aestivum and Hordeum lechleri suggest that the pathway is of monophyletic origin in the Poaceae. The branchpoint from the primary metabolism (Bx1 gene) can be traced back to duplication and functionalisation of the alpha-subunit of tryptophan synthase (TSA). Modification of the intermediates by consecutive hydroxylation is catalysed by members of a cytochrome P450 enzyme subfamily (Bx2–Bx5). Glucosylation by an UDP-glucosyltransferase (UGT, Bx8, Bx9) is essential for the reduction of autotoxicity of the benzoxazinoids. In some species 2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside (DIBOA-glc) is further modified by the 2-oxoglutarate-dependent dioxygenase BX6 and the O-methyltransferase BX7. In the dicots Aphelandra squarrosa, Consolida orientalis, and Lamium galeobdolon, benzoxazinoid biosynthesis is analogously organised: The branchpoint is established by a homolog of TSA, P450 enzymes catalyse hydroxylations and at least the first hydroxylation reaction is identical in dicots and Poaceae, the toxic aglucon is glucosylated by an UGT. Functionally, TSA and BX1 are indole-glycerolphosphate lyases (IGLs). Igl genes seem to be generally duplicated in angiosperms. Modelling and biochemical characterisation of IGLs reveal that the catalytic properties of the enzyme can easily be modified by mutation. Independent evolution can be assumed for the BX1 function in dicots and Poaceae.     

The evolutionary history of the first three enzymes in pyrimidine biosynthesis

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD

1 Abbreviations: CAD, trifunctional protein catalyzing the first three steps of de novo pyrimidine biosynthesis in higher eukaryotes; CPS, carbamyl phosphate synthetase domain; CPSase, carbamyl phosphate synthetase activity; ATC, aspartate transcarbamylase domain; ATCase, aspartate transcarbamylase activity; DHO, dihydroorotase domain; DHOase, dihydroorotase activity; GLN, glutaminase subdomain or subunit of carbamyl phosphate synthetase, GL Nase, glutaminase activity; SYN, synthetase subdomain or subunit of carbamyl phosphate synthetase; SYNase, synthetase activity.

carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.     

Piezotolerance as a metabolic engineering tool for the biosynthesis of natural products

Thermodynamically, high-pressure (>10's of MPa) has a potentially vastly superior effect on reactions and their rates within metabolic processes than temperature. Thus, it might be expected that changes in the pressure experienced by living organisms would have effects on the products of their metabolism. To examine the potential for modification of metabolic pathways based on thermodynamic principles we have performed simple molecular dynamics simulations, in vacuo and in aquo on the metabolites synthesized by recombinant polyketide synthases (PKS). We were able to determine, in this in silico study, the volume changes associated with each reaction step along the parallel PKS pathways. Results indicate the importance of explicitly including the solvent in the simulations. Furthermore, the addition of solvent and high pressure reveals that high pressure may have a beneficial effect on certain pathways over others. Thus, the future looks bright for pressure driven novel secondary metabolite discoveries, and their sustained and efficient production via metabolic engineering.     

The evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine

Ergothioneine (EGT) is a histidine betaine derivative that exhibits antioxidant action in humans. EGT is primarily synthesized by fungal species and a number of bacterial species. A five-gene cluster (egtA, egtB, egtC, egtD & egtE) responsible for EGT production in Mycobacteria smegmatis has recently been identified. The first fungal biosynthetic EGT gene (NcEgt-1) has also been identified in Neurospora crassa. NcEgt-1 contains domains similar to those found in M. smegmatis egtB and egtD. EGT is biomembrane impermeable. Here we inferred the evolutionary history of the EGT cluster in prokaryotes as well as examining the phyletic distribution of Egt-1 in the fungal kingdom. A genomic survey of 2509 prokaryotes showed that the five-gene EGT cluster is only found in the Actinobacteria. Our survey identified more than 400 diverse prokaryotes that contain genetically linked orthologs of egtB and egtD. Phylogenetic analyses of Egt proteins show a complex evolutionary history and multiple incidences of horizontal gene transfer. Our analysis also identified two independent incidences of a fusion event of egtB and egtD in bacterial species. A genomic survey of over 100 fungal genomes shows that Egt-1 is found in all fungal phyla, except species that belong to the Saccharomycotina subphylum. This analysis provides a comprehensive analysis of the distribution of the key genes involved in the synthesis of EGT in prokaryotes and fungi. Our phylogenetic inferences illuminate the complex evolutionary history of the genes involved in EGT synthesis in prokaryotes. The potential to synthesize EGT is a fungal trait except for species belonging to the Saccharomycotina subphylum.     

Assessing the prospects for a return of organisms in evolutionary biology

An argument has been raised from various perspectives against the Modern Synthesis (MS) in the past two decades: it has forgotten organisms. Niche construction theorists (Odling-Smee et al. 2003), developmental biologists like West-Eberhard (2003) and Evo-Devo elaborated various views which concur on a rehabilitation of the explanatory role of organisms, formerly neglected by an evolutionary science mostly centered on genes. This paper aims at assessing such criticisms by unraveling the specific arguments they use and evaluating how empirical findings may support them. In the first section, I review the usual critiques about the way MS treats organisms and show that the organisms-concerned critique is multifaceted, and I use the controversy about units of selection in order to show that purely conceptual and empirical arguments have been mixed up when organisms were concerned. In the second section, I consider successively the challenges raised to evolutionary MS by structuralist biologists and then the developmentalist challenge mostly raised by Evo-Devo. I distinguish what is purely conceptual among those criticisms and what mostly relies on recent empirical findings about genome activation, inheritance, and epigenetics. The last section discusses another program in MS, namely "evolutionary transitions" research, as enquiry into the emergence of organisms.     

The role of gene fusions in the evolution of metabolic pathways: the histidine biosynthesis case

Background

Histidine biosynthesis is one of the best characterized anabolic pathways. There is a large body of genetic and biochemical information available, including operon structure, gene expression, and increasingly larger sequence databases. For over forty years this pathway has been the subject of extensive studies, mainly in Escherichia coli and Salmonella enterica, in both of which details of histidine biosynthesis appear to be identical. In these two enterobacteria the pathway is unbranched, includes a number of unusual reactions, and consists of nine intermediates; his genes are arranged in a compact operon (hisGDC [NB]HAF [IE]), with three of them (hisNB, hisD and hisIE) coding for bifunctional enzymes. We performed a detailed analysis of his gene fusions in available genomes to understand the role of gene fusions in shaping this pathway.

Results

The analysis of HisA structures revealed that several gene elongation events are at the root of this protein family: internal duplication have been identified by structural superposition of the modules composing the TIM-barrel protein.Several his gene fusions happened in distinct taxonomic lineages; hisNB originated within γ-proteobacteria and after its appearance it was transferred to Campylobacter species (ε-proteobacteria) and to some Bacteria belonging to the CFB group. The transfer involved the entire his operon. The hisIE gene fusion was found in several taxonomic lineages and our results suggest that it probably happened several times in distinct lineages.Gene fusions involving hisIE and hisD genes (HIS4) and hisH and hisF genes (HIS7) took place in the Eukarya domain; the latter has been transferred to some δ-proteobacteria.

Conclusion

Gene duplication is the most widely known mechanism responsible for the origin and evolution of metabolic pathways; however, several other mechanisms might concur in the process of pathway assembly and gene fusion appeared to be one of the most important and common.

     

On the origins of cells: a hypothesis for the evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes,and from prokaryotes to nucleated cells

All life is organized as cells. Physical compartmentation from the environment and self-organization of self-contained redox reactions are the most conserved attributes of living things, hence inorganic matter with such attributes would be life's most likely forebear. We propose that life evolved in structured iron monosulphide precipitates in a seepage site hydrothermal mound at a redox, pH and temperature gradient between sulphide-rich hydrothermal fluid and iron(II)-containing waters of the Hadean ocean floor. The naturally arising, three-dimensional compartmentation observed within fossilized seepage-site metal sulphide precipitates indicates that these inorganic compartments were the precursors of cell walls and membranes found in free-living prokaryotes. The known capability of FeS and NiS to catalyse the synthesis of the acetyl-methylsulphide from carbon monoxide and methylsulphide, constituents of hydrothermal fluid, indicates that pre-biotic syntheses occurred at the inner surfaces of these metal-sulphide-walled compartments, which furthermore restrained reacted products from diffusion into the ocean, providing sufficient concentrations of reactants to forge the transition from geochemistry to biochemistry. The chemistry of what is known as the RNA-world could have taken place within these naturally forming, catalyticwalled compartments to give rise to replicating systems. Sufficient concentrations of precursors to support replication would have been synthesized in situ geochemically and biogeochemically, with FeS (and NiS) centres playing the central catalytic role. The universal ancestor we infer was not a free-living cell, but rather was confined to the naturally chemiosmotic, FeS compartments within which the synthesis of its constituents occurred. The first free-living cells are suggested to have been eubacterial and archaebacterial chemoautotrophs that emerged more than 3.8 Gyr ago from their inorganic confines. We propose that the emergence of these prokaryotic lineages from inorganic confines occurred independently, facilitated by the independent origins of membrane-lipid biosynthesis: isoprenoid ether membranes in the archaebacterial and fatty acid ester membranes in the eubacterial lineage. The eukaryotes, all of which are ancestrally heterotrophs and possess eubacterial lipids, are suggested to have arisen ca. 2 Gyr ago through symbiosis involving an autotrophic archaebacterial host and a heterotrophic eubacterial symbiont, the common ancestor of mitochondria and hydrogenosomes. The attributes shared by all prokaryotes are viewed as inheritances from their confined universal ancestor. The attributes that distinguish eubacteria and archaebacteria, yet are uniform within the groups, are viewed as relics of their phase of differentiation after divergence from the non-free-living universal ancestor and before the origin of the free-living chemoautotrophic lifestyle. The attributes shared by eukaryotes with eubacteria and archaebacteria, respectively, are viewed as inheritances via symbiosis. The attributes unique to eukaryotes are viewed as inventions specific to their lineage. The origin of the eukaryotic endomembrane system and nuclear membrane are suggested to be the fortuitous result of the expression of genes for eubacterial membrane lipid synthesis by an archaebacterial genetic apparatus in a compartment that was not fully prepared to accommodate such compounds, resulting in vesicles of eubacterial lipids that accumulated in the cytosol around their site of synthesis. Under these premises, the most ancient divide in the living world is that between eubacteria and archaebacteria, yet the steepest evolutionary grade is that between prokaryotes and eukaryotes.     

Retroposed elements as archives for the evolutionary history of placental mammals

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.     

New insights into the evolutionary history of photoreceptor cells

Although the common descent of all life has been widely accepted since Darwin's time, new research occasionally provides us with arresting reminders of the unity of evolutionary history. Recent papers by Arendt et al. and Panda et al. provide one such reminder. They illustrate that the two classes of animal photoreceptors, ciliary and rhabdomeric photoreceptors, are likely to share an ancient common ancestor and have been evolving in parallel since their duplication over 600 million years ago.     

Selfish operons: the evolutionary impact of gene clustering in prokaryotes and eukaryotes

The Selfish Operon Model postulates that the organization of bacterial genes into operons is beneficial to the constituent genes in that proximity allows horizontal cotransfer of all genes required for a selectable phenotype; eukaryotic operons formed for very different reasons. Horizontal transfer of selfish operons most probably promotes bacterial diversification.     

Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue

Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the "two-step branching and improper branch clearing model" is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, alpha-1,6-branch formation and the subsequent alpha-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster.