Evolution of structure and function in Phenylalanine Hydroxylase. With the regulatory properties in sight

In the post-genomic era, an idea of how similar the genomes of different species actually are is on the horizon. Less than 10 years ago, the human genome was estimated to encode 100000 genes. That was an overestimation, as the real number of human genes is 20000-25000. Most genes are expressed as proteins. The 3D structure of a protein is more conserved than its sequence, and therefore the structural context of protein and gene evolution must not be forgotten. By its structure, the protein can propagate its function. In the early 90’s the estimated number of different protein structure classes, so called folds, was predicted to be about 10000. Today there are slightly above 1000 folds and the discovery of new folds has leveled off, despite an increase in the number of protein structures that have been solved over the last few years. Indeed, some folds are used for more than one function, and found in various functional contexts. Then, if the many components are so similar, how is the biological species divergence from same component genomes achieved? One way to study biological diversity is by dividing it into its smaller components, e.g. by studying protein or gene family evolution. Here the evolution and regulation of the aromatic amino acid hydroxylase (AAAHs) have been under examination. This gene family encodes the proteins phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH). These enzymes are highly physiologically important. PAH, expressed in liver, regulates the homeostasis of L-Phe by hydroxylating it into L-Tyr. TH, expressed in the central nervous system, hydroxylates L-Tyr into L-Dopa. L-Dopa is part of two important pathways i) melanogenesis and ii) dopamine production. In humans, dysfunctions in PAH that cause elevated L-Phe concentration can result in phenylketonuria (PKU). Untreated PKU results in neurological damage. TPH produces a precursor of serotonin from LTrp. The end products of these enzymes are neurotransmitters and hormones with increasingly important functions, from e.g. amoeba to nematode to man. As PAH has evolved in mammals its regulation has become increasingly sophisticated, e.g. homotropic positive cooperativity that shifts the conformational equilibrium from dimeric to tetrameric is seen in the mammalian lineage. Nematode PAH is devoid of positive cooperativity, but resembles the tetrameric high-affinity and high-activity mammalian PAH. TH and TPH are always tetrameric and not allosterically regulated. Each AAAH subunit has a regulatory domain, a catalytic domain, and an oligomerization domain. The promotion of positive cooperativity in PAH has been investigated by comparing mammalian PAH to nematode PAH. The low-affinity and low-activity dimer as well as the high-affinity and high-activity tetramer of PAH were modeled. Sequence analysis on a nematode sequence cluster and a mammalian sequence cluster identified sites with high probability of being involved in functional divergence, e.g. change in regulation. Residue specific electrostatic interaction energies were calculated for all ionizible residues in the models. In general, we note important differences in the substrate binding pocket that aids to explain why the active site in nematode PAH is less dynamic than in mammalian PAH. Our results suggest a pathway for the positive cooperativity from one active site to another, involving various predicted hinge regions from human PAH, where we find the nematode PAH more rigid. The regulatory domain in PAH is part of the ACT domain family. The ACT domains are frequently found regulating metabolic enzymes in an allosteric manner. The allosteric effector is often an amino acid that binds to an interface formed by two ACT domains. No contacts are formed between two ACT domains and the stoichiometry of binding is 1:1 for L-Phe in PAH. Therefore the allosteric effect must originate in the active site when the substrate binds. An alternative pathway for aromatic amino acid biosynthesis is present in e.g. plants and bacteria. This pathway has an L-Phe binding ACT domain, which is homologous to the ACT domain in AAAH. The L-Phe binding motif in this domain is also conserved in PAH. A comparative structural analysis of this area shows why L-Phe may not bind in the AAAH regulatory domain and also indicates why it has remained. The ACT domain has an abundant fold, a superfold. A structural approach was used to identify more potential ACT domains to gain further insights to the functional properties that this domain could perform in general, and in PAH in particular. Here we note e.g. two interesting potential domain families that could be homologous to the ACT domain, namely the GlnB-like domains and heavy metal binding domains. The phylogeny of the AAAH family has not been resolved earlier given the lack of a suitable outgroup. As more genome sequences became available, we identified an outgroup candidate and had it experimentally characterized. The phylogeny was resolved, the ancestral function determined, and by comparing the chromosomal gene locations the order of events in AAAH evolution was envisioned

A systematic search for positive selection in higher plants (Embryophytes)

BACKGROUND: Previously, a database characterizing examples of Embryophyte gene family lineages showing evidence of positive selection was reported. Of the gene family trees, 138 Embryophyte branches showed Ka/Ks>>1 and are candidates for functional adaptation. The database and these examples have now been studied in further detail to better understand the molecular basis for plant genome evolution. RESULTS: Neutral modeling showed an excess of positive and/or negative selection in the database over a neutral expectation centered on the mean Ka/Ks ratio. Out of 673 families with assigned structures, 490 have at least one branch with Ka/Ks >>1 in a region of the protein, enabling a picture of selective pressures delineated by protein structure. Most gene families allowed reconstruction back to the last common ancestor of flowering plants (Magnoliophytes) without saturation of 4- fold degenerate codon position. Positive selection occurred in a wide variety of gene families with different functions, including in the self incompatibility locus, in defense against pathogens, in embryogenesis, in cold acclimation, and in electrontransport. Structurally, selective pressures were similar between alpha-helices and beta- sheets, but were less negative and more variant on the surface and away from the hydrophobic core. CONCLUSION: Positive selection was detected statistically significantly in a small and nonrandom minority of gene families in a systematic analysis of embryophyte gene families. More sensitive methods increased the level of positive selection that was detected and presented a structural basis for the role of positive selection in plant genomes

Avoiding Regions Symptomatic of Conformational and Functional Flexibility to Identify Antiviral Targets in Current and Future Coronaviruses

Within the last 15 years, two related coronaviruses (Severe Acute Respiratory Syndrome [SARS]-CoV and Middle East Respiratory Syndrome [MERS]-CoV) expanded their host range to include humans, with increased virulence in their new host. Coronaviruses were recently found to have little intrinsic disorder compared with many other virus families. Because intrinsically disordered regions have been proposed to be important for rewiring interactions between virus and host, we investigated the conservation of intrinsic disorder and secondary structure in coronaviruses in an evolutionary context. We found that regions of intrinsic disorder are rarely conserved among different coronavirus protein families, with the primary exception of the nucleocapsid. Also, secondary structure predictions are only conserved across 50–80% of sites for most protein families, with the implication that 20–50% of sites do not have conserved secondary structure prediction. Furthermore, nonconserved structure sites are significantly less constrained in sequence divergence than either sites conserved in the secondary structure or sites conserved in loop. Avoiding regions symptomatic of conformational flexibility such as disordered sites and sites with nonconserved secondary structure to identify potential broad-specificity antiviral targets, only one sequence motif (five residues or longer) remains from the \u3e10,000 starting sites across all coronaviruses in this study. The identified sequence motif is found within the nonstructural protein (NSP) 12 and constitutes an antiviral target potentially effective against the present day and future coronaviruses. On shorter evolutionary timescales, the SARS and MERS clades have more sequence motifs fulfilling the criteria applied. Interestingly, many motifs map to NSP12 making this a prime target for coronavirus antivirals

Subfunctionalization of duplicated genes as a transition state to neofunctionalization

BACKGROUND: Gene duplication has been suggested to be an important process in the generation of evolutionary novelty. Neofunctionalization, as an adaptive process where one copy mutates into a function that was not present in the pre-duplication gene, is one mechanism that can lead to the retention of both copies. More recently, subfunctionalization, as a neutral process where the two copies partition the ancestral function, has been proposed as an alternative mechanism driving duplicate gene retention in organisms with small effective population sizes. The relative importance of these two processes is unclear. RESULTS: A set of lattice model genes that fold and bind to two peptide ligands with overlapping binding pockets, but not a third ligand present in the cell was designed. Each gene was duplicated in a model haploid species with a small constant population size and no recombination. One set of models allowed subfunctionalization of binding events following duplication, while another set did not allow subfunctionalization. Modeling under such conditions suggests that subfunctionalization plays an important role, but as a transition state to neofunctionalization rather than as a terminal fate of duplicated genes. There is no apparent selective pressure to maintain redundancy. CONCLUSION: Subfunctionalization results in an increase in the preservation of duplicated gene copies, including those that are neofunctionalized, but never represents a substantial fraction of duplicate gene copies at any evolutionary time point and ultimately leads to neofunctionalization of those preserved copies. This conclusion also may reflect changes in gene function after duplication with time in real genomes

Apoptosis-inducing natural products found in utero during murine pregnancy

AbstractBackground: Hormones, lipids, vitamins and other biologically active small molecules can be removed from animal tissues by extraction with organic solvents. These compounds can have dramatic effects on cultured cells and the characterization of such compounds can lead to the discovery of new functions for known molecules, or even to the discovery of previously unknown compounds.Results: Organic-soluble compounds in 17.5-day-old mouse embryos were removed with tert-butylmethylether and found to induce apoptosis in T-antigen-transformed Jurkat T cells. These embryonic extracts were fractionated and their apoptosis-inducing components were identified as a mixture of polyunsaturated fatty acids, including arachidonic, docosatetraenoic and docosahexaenoic acids. Docosatetraenoic acid was the most potent apoptosis inducer with an effective dose (ED50) of 30 μM.Conclusions: A family of polyunsaturated fatty acids is shown to be abundant in utero during pregnancy. Members of this family are able to induce cleavage of poly(ADP)ribose polymerase, and ultimately to induce apoptosis, in T-antigen-transformed Jurkat T cells. Free radical scavengers, including phenol and benzyl alcohol, block the apoptosis-inducing properties of these polyunsaturated fatty acids; this is consistent with a lipid peroxidation mechanism involving formation of hydroperoxy fatty acids

Visualising very large phylogenetic trees in three dimensional hyperbolic space

BACKGROUND: Common existing phylogenetic tree visualisation tools are not able to display readable trees with more than a few thousand nodes. These existing methodologies are based in two dimensional space. RESULTS: We introduce the idea of visualising phylogenetic trees in three dimensional hyperbolic space with the Walrus graph visualisation tool and have developed a conversion tool that enables the conversion of standard phylogenetic tree formats to Walrus' format. With Walrus, it becomes possible to visualise and navigate phylogenetic trees with more than 100,000 nodes. CONCLUSION: Walrus enables desktop visualisation of very large phylogenetic trees in 3 dimensional hyperbolic space. This application is potentially useful for visualisation of the tree of life and for functional genomics derivatives, like The Adaptive Evolution Database (TAED)