Allosteric regulation of glutamate dehydrogenase deamination activity.

Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia (HI/HA) syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of allosteric network behind GDH regulation, identifications of allosteric factors and associated mechanisms are paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our structural analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of the inhibitor GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH-bound GDH activates a triangular allosteric network, interlinking the inhibitor with regulatory and catalytic sites. This allostery produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. In silico mutagenesis examinations further underpin the molecular basis of HI/HA dominant mutations and consequent over-activity of GDH through alteration of this allosteric communication network. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.

Stoichio-Metagenomics of Ocean Waters: A Molecular Evolution Approach to Trace the Dynamics of Nitrogen Conservation in Natural Communities.

Nitrogen is crucially limiting in ocean surface waters, and its availability varies substantially with coastal regions typically richer in nutrients than open oceans. In a biological stoichiometry framework, a parsimonious strategy of nitrogen allocation predicts nitrogen content of proteins to be lower in communities adapted to open ocean than to coastal regions. To test this hypothesis we have directly interrogated marine microbial communities, using a series of metagenomics datasets with a broad geographical distribution from the Global Ocean Sampling Expedition. Analyzing over 20 million proteins, we document a ubiquitous signal of nitrogen conservation in open ocean communities, both in membrane and non-membrane proteins. Efficient nitrogen allocation is expected to specifically target proteins that are expressed at high rate in response to nitrogen starvation. Furthermore, in order to preserve protein functional efficiency, economic nitrogen allocation is predicted to target primarily the least functionally constrained regions of proteins. Contrasting the NtcA-induced pathway, typically up-regulated in response to nitrogen starvation, with the arginine anabolic pathway, which is instead up-regulated in response to nitrogen abundance, we show how both these predictions are fulfilled. Using evolutionary rates as an informative proxy of functional constraints, we show that variation in nitrogen allocation between open ocean and coastal communities is primarily localized in the least functionally constrained regions of the genes triggered by NtcA. As expected, such a pattern is not detectable in the genes involved in the arginine anabolic pathway. These results directly link environmental nitrogen availability to different adaptive strategies of genome evolution, and emphasize the relevance of the material costs of evolutionary change in natural ecosystems.

Organization of biogeochemical nitrogen pathways with switch-like adjustment in fluctuating soil redox conditions.

Nitrogen is cycled throughout ecosystems by a suite of biogeochemical processes. The high complexity of the nitrogen cycle resides in an intricate interplay between reversible biochemical pathways alternatively and specifically activated in response to diverse environmental cues. Despite aggressive research, how the fundamental nitrogen biochemical processes are assembled and maintained in fluctuating soil redox conditions remains elusive. Here, we address this question using a kinetic modelling approach coupled with dynamical systems theory and microbial genomics. We show that alternative biochemical pathways play a key role in keeping nitrogen conversion and conservation properties invariant in fluctuating environments. Our results indicate that the biochemical network holds inherent adaptive capacity to stabilize ammonium and nitrate availability, and that the bistability in the formation of ammonium is linked to the transient upregulation of the amo-hao mediated nitrification pathway. The bistability is maintained by a pair of complementary subsystems acting as either source or sink type systems in response to soil redox fluctuations. It is further shown how elevated anthropogenic pressure has the potential to break down the stability of the system, altering substantially ammonium and nitrate availability in the soil, with dramatic effects on biodiversity.

Robust regulation of hepatic pericentral amination by glutamate dehydrogenase kinetics.

Impaired glutamate dehydrogenase (GDH) sensitivity to its inhibitors causes excessive insulin secretion by pancreatic beta-cells and defective ammonia metabolism in the liver. These symptoms are commonly associated with hyperinsulinism/hyperammonemia syndrome (HI/HA), which causes recurrent hypoglycaemia in early infancy. Hepatic localization of GDH amination and deamination activities linked with the urea cycle is known to be involved in ammonia metabolism and detoxification. Although deamination activities of hepatic GDH in the periportal zones of liver lobules and its connection to the urea cycle have been exhaustively investigated, physiological roles of GDH amination activity observed at pericentral zones have often been overlooked. Using kinetic modelling approaches, here we report a new role for hepatic GDH amination kinetics in maintaining ammonia homeostasis under an excess intrahepatocyte input of ammonium. We have shown that α-ketoglutarate substrate inhibition kinetics of GDH, which include both random and obligatory ordered association/dissociation reactions, robustly control the ratio between glutamate and ammonium under a wide range of intracellular substrate variation. Dysregulation of this activity under pericentral nitrogen insufficiency contributes to the breaking down of ammonia homeostasis and thereby can significantly affect HI/HA syndrome.

GRASP [Genomic Resource Access for Stoichioproteomics]: comparative explorations of the atomic content of 12 Drosophila proteomes.

BACKGROUND: "Stoichioproteomics" relates the elemental composition of proteins and proteomes to variation in the physiological and ecological environment. To help harness and explore the wealth of hypotheses made possible under this framework, we introduce GRASP (http://www.graspdb.net), a public bioinformatic knowledgebase containing information on the frequencies of 20 amino acids and atomic composition of their side chains. GRASP integrates comparative protein composition data with annotation data from multiple public databases. Currently, GRASP includes information on proteins of 12 sequenced Drosophila (fruit fly) proteomes, which will be expanded to include increasingly diverse organisms over time. In this paper we illustrate the potential of GRASP for testing stoichioproteomic hypotheses by conducting an exploratory investigation into the composition of 12 Drosophila proteomes, testing the prediction that protein atomic content is associated with species ecology and with protein expression levels. RESULTS: Elements varied predictably along multivariate axes. Species were broadly similar, with the D. willistoni proteome a clear outlier. As expected, individual protein atomic content within proteomes was influenced by protein function and amino acid biochemistry. Evolution in elemental composition across the phylogeny followed less predictable patterns, but was associated with broad ecological variation in diet. Using expression data available for D. melanogaster, we found evidence consistent with selection for efficient usage of elements within the proteome: as expected, nitrogen content was reduced in highly expressed proteins in most tissues, most strongly in the gut, where nutrients are assimilated, and least strongly in the germline. CONCLUSIONS: The patterns identified here using GRASP provide a foundation on which to base future research into the evolution of atomic composition in Drosophila and other taxa.

Stoichiogenomics: the evolutionary ecology of macromolecular elemental composition.

The new field of 'stoichiogenomics' integrates evolution, ecology and bioinformatics to reveal surprising patterns of the differential usage of key elements [e.g. nitrogen (N)] in proteins and nucleic acids. Because the canonical amino acids as well as nucleotides differ in element counts, natural selection owing to limited element supplies might bias monomer usage to reduce element costs. For example, proteins that respond to N limitation in microbes use a lower proportion of N-rich amino acids, whereas proteome- and transcriptome-wide element contents differ significantly for plants as compared with animals, probably because of the differential severity of element limitations. In this review, we show that with these findings, new directions for future investigations are emerging, particularly via the increasing availability of diverse metagenomic and metatranscriptomic data sets.

Assessing the influence of adjacent gene orientation on the evolution of gene upstream regions in Arabidopsis thaliana.

The orientation of flanking genes may influence the evolution of intergenic regions in which cis-regulatory elements are likely to be located: divergently transcribed genes share their 5' regions, resulting either in smaller "private" spaces or in overlapping regulatory elements. Thus, upstream sequences of divergently transcribed genes (bi-directional upstream regions, or URs) may be more constrained than those of uni-directional gene pairs. We investigated this effect by analyzing nucleotide variation segregating within and between Arabidopsis species. Compared to uni-directional URs, bi-directional URs indeed display lower population mutation rate, as well as more low-frequency polymorphisms. Furthermore, we find that bi-directional regions undergo selection for the maintenance of intergenic distance. Altogether, however, we observe considerable variation in evolutionary rates, with putative signatures of selection on two uni-directional upstream regions.

Signatures of nitrogen limitation in the elemental composition of the proteins involved in the metabolic apparatus.

Nitrogen (N) is a fundamental component of nucleotides and amino acids and is often a limiting nutrient in natural ecosystems. Thus, study of the N content of biomolecules may establish important connections between ecology and genomics. However, while significant differences in the elemental composition of whole organisms are well documented, how the flux of nutrients in the cell has shaped the evolution of different cellular processes remains poorly understood. By examining the elemental composition of major functional classes of proteins in four multicellular eukaryotic model organisms, we find that the catabolic machinery shows substantially lower N content than the anabolic machinery and the rest of the proteome. This pattern suggests that ecological selection for N conservation specifically targets cellular components that are highly expressed in response to nutrient limitation. We propose that the RNA component of the anabolic machineries is the mechanistic force driving the elemental imbalance we found, and that RNA functions as an intracellular nutrient reservoir that is degraded and recycled during starvation periods. A comparison of the elemental composition of the anabolic and catabolic machineries in species that have experienced different levels of N limitation in their evolutionary history (animals versus plants) suggests that selection for N conservation has preferentially targeted the catabolic machineries of plants, resulting in a lower N content of the proteins involved in their catabolic processes. These findings link the composition of major cellular components to the environmental factors that trigger the activation of those components, suggesting that resource availability has constrained the atomic composition and the molecular architecture of the biotic processes that enable cells to respond to reduced nutrient availability.

Ecological nitrogen limitation shapes the DNA composition of plant genomes.

Phenotypes and behaviors respond to resource constraints via adaptation, but the influence of ecological limitations on the composition of eukaryotic genomes is still unclear. We trace connections between plant ecology and genomes through their elemental composition. Inorganic sources of nitrogen (N) are severely limiting to plants in natural ecosystems. This constraint would favor the use of N-poor nucleotides in plant genomes. We show that the transcribed segments of undomesticated plant genomes are the most N poor, with genomes and proteomes bearing signatures of N limitation. Consistent with the predictions of natural selection for N conservation, the precursors of transcriptome show the greatest deviations from Chargaff's second parity rule. Furthermore, crops show higher N contents than undomesticated plants, likely due to the relaxation of natural selection owing to the use of N-rich fertilizers. These findings indicate a fundamental role of N limitation in the evolution of plant genomes, and they link the genomes with the ecosystem context within which biota evolve.

Nullomers: really a matter of natural selection?

BACKGROUND: Nullomers are short DNA sequences that are absent from the genomes of humans and other species. Assuming that nullomers are the signatures of natural selection against deleterious sequences in humans, the use of nullomers in drug target identification, pesticide development, environmental monitoring, and forensic applications has been envisioned. RESULTS: Here, we show that the hypermutability of CpG dinucleotides, rather than the natural selection against the nullomer sequences, is likely the reason for the phenomenal event of short sequence motifs becoming nullomers. Furthermore, many reported human nullomers differ by only one nucleotide, which reinforces the role of mutation in the evolution of the constellation of nullomers in populations and species. The known nullomers in chimpanzee, cow, dog, and mouse genomes show patterns that are consistent with those seen in humans. CONCLUSIONS: The role of mutations, instead of selection, in generating nullomers cast doubt on the utility of nullomers in many envisioned applications, because of their dependence on the role of lethal selection on the origin of nullomers.

Adaptive walks toward a moving optimum.

We investigate how the dynamics and outcomes of adaptation by natural selection are affected by environmental stability by simulating adaptive walks in response to an environmental change of fixed magnitude but variable speed. Here we consider monomorphic lineages that adapt by the sequential fixation of beneficial mutations. This is modeled by selecting short RNA sequences for folding stability and secondary structure conservation at increasing temperatures. Using short RNA sequences allows us to describe adaptive outcomes in terms of genotype (sequence) and phenotype (secondary structure) and to follow the dynamics of fitness increase. We find that slower rates of environmental change affect the dynamics of adaptive walks by reducing the fitness effect of fixed beneficial mutations, as well as by increasing the range of time in which the substitutions of largest effect are likely to occur. In addition, adaptation to slower rates of environmental change results in fitter endpoints with fewer possible end phenotypes relative to lineages that adapt to a sudden change. This suggests that care should be taken when experiments using sudden environmental changes are used to make predictions about adaptive responses to gradual change.

Oxygen content of transmembrane proteins over macroevolutionary time scales.

We observe that the time of appearance of cellular compartmentalization correlates with atmospheric oxygen concentration. To explore this correlation, we predict and characterize the topology of all transmembrane proteins in 19 taxa and correlate differences in topology with historical atmospheric oxygen concentrations. Here we show that transmembrane proteins, individually and as a group, were probably selectively excluding oxygen in ancient ancestral taxa, and that this constraint decreased over time when atmospheric oxygen levels rose. As this constraint decreased, the size and number of communication-related transmembrane proteins increased. We suggest the hypothesis that atmospheric oxygen concentrations affected the timing of the evolution of cellular compartmentalization by constraining the size of domains necessary for communication across membranes.