Functional characterization of Arabidopsis thaliana transthyretin-like protein

<p>Abstract</p> <p>Background</p> <p><it>Arabidopsis thaliana </it>transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.</p> <p>Results</p> <p>The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities <it>in vitro</it>, in the full-length protein.</p> <p>Conclusions</p> <p>The <it>Arabidopsis thaliana </it>transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed <it>in vitro</it>.</p

An estimate of the number of tropical tree species

The high species richness of tropical forests has long been recognized, yet there remains substantial uncertainty regarding the actual number of tropical tree species. Using a pantropical tree inventory database from closed canopy forests, consisting of 657,630 trees belonging to 11,371 species, we use a fitted value of Fisher’s alpha and an approximate pantropical stem total to estimate the minimum number of tropical forest tree species to fall between ∼40,000 and ∼53,000, i.e. at the high end of previous estimates. Contrary to common assumption, the Indo-Pacific region was found to be as species-rich as the Neotropics, with both regions having a minimum of ∼19,000–25,000 tree species. Continental Africa is relatively depauperate with a minimum of ∼4,500–6,000 tree species. Very few species are shared among the African, American, and the Indo-Pacific regions. We provide a methodological framework for estimating species richness in trees that may help refine species richness estimates of tree-dependent taxa

Phylogenetic classification of the world's tropical forests

Knowledge about the biogeographic affinities of the world’s tropical forests helps to better understand regional differences in forest structure, diversity, composition, and dynamics. Such understanding will enable anticipation of region-specific responses to global environmental change. Modern phylogenies, in combination with broad coverage of species inventory data, now allow for global biogeographic analyses that take species evolutionary distance into account. Here we present a classification of the world’s tropical forests based on their phylogenetic similarity. We identify five principal floristic regions and their floristic relationships: (i) Indo-Pacific, (ii) Subtropical, (iii) African, (iv) American, and (v) Dry forests. Our results do not support the traditional neo- versus paleotropical forest division but instead separate the combined American and African forests from their Indo-Pacific counterparts. We also find indications for the existence of a global dry forest region, with representatives in America, Africa, Madagascar, and India. Additionally, a northern-hemisphere Subtropical forest region was identified with representatives in Asia and America, providing support for a link between Asian and American northern-hemisphere forests.</p

Exploring O₂ Diffusion in A-Type Cytochrome <i>c</i> Oxidases: Molecular Dynamics Simulations Uncover Two Alternative Channels towards the Binuclear Site

<div><p>Cytochrome <i>c</i> oxidases (C<i>c</i>oxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O₂) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type C<i>c</i>ox family, little is known about the channel(s) used by O₂ to travel from the solvent/membrane to the heme <i>a₃</i>-Cu_B binuclear center (BNC). Moreover, the identification of all possible O₂ channels as well as the atomic details of O₂ diffusion is essential for the understanding of the working mechanisms of the A-type C<i>c</i>ox. In this work, we determined the O₂ distribution within C<i>c</i>ox from <i>Rhodobacter sphaeroides</i>, in the fully reduced state, in order to identify and characterize all the putative O₂ channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O₂ molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O₂ inside C<i>c</i>ox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O₂ to reach the BNC. Both alternative O₂ channels start in the membrane spanning region and terminate close to Y288_I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this C<i>c</i>ox, the most likely (energetically preferred) routes for O₂ to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.</p></div

Free energy barriers experienced by O₂.

<p>Free energy barriers experienced by O₂ when moving from the membrane region to the BNC, along Channel 1 (A), Channel 2 (B) and Channel 3 (C). For details related to the errors calculation see the data analysis section of the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004010#s3" target="_blank">Materials and Methods</a>. The “*” in the fig. indicates the transitions for which the errors could not be calculated. In Fig. A, the black lines correspond to Channel 1, which starts between helices 5 and 8 of subunit I, whereas the orange lines correspond to the second entrance point located between helices 11 and 13 of subunit I. In A and C, the dashed lines correspond to alternative routes for O₂ inside the same channel (for example in Fig. C, O₂ can move directly from M₁₄ to M₁₆, or it can go from M₁₄ to M₁₅ and only then to M₁₆). The numbers inside the plots on top of the transition states indicate the free energy barriers experienced by O₂ when moving from the membrane in the direction of the BNC (i.e. between the different minima on their immediate left and the transition states in question).</p

O₂ free energy landscape obtained from the ILS calculations and lowest free energy pathways obtained from the ILS energy landscape.

<p><b>A-</b>O₂ free energy landscape obtained from the ILS calculations. Two isosurface contours are shown, with free energy levels at -5 (blue inner contour) and 5 (cyan contour) kJ·mol⁻¹. <b>B-</b> Comparison of the O₂ free energy landscape obtained from the ILS calculations (blue maps) and high-affinity regions from the O₂ explicit MD simulations (red maps). The probability density contours at 0.00015 Å⁻³ are represented as a red isosurface. <b>C-</b> O₂ lowest free energy pathways obtained from the ILS energy landscape. The spheres represent the local free energy minima while the tubes connecting the minima represent the pathways. The size of the spheres representing the local energy minima scales linearly along the displayed free energy range (small spheres indicates high free energy while large spheres indicate low free energy). The diameter of the pathways scales linearly with the free energy and thus with the oxygen affinity (thinner radius represent high free energies and low oxygen affinity). The free energy at the minima and along the pathways follows the same color scale. The channel 1 coincides with the channel inferred from the X-ray data <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004010#pcbi.1004010-Tsukihara1" target="_blank">[7]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004010#pcbi.1004010-SvenssonEk1" target="_blank">[13]</a> whereas the channel 2 and channel 3 are alternative routes to reach the BNC.</p

Overview of the O₂ lowest free energy pathways obtained from the ILS free energy landscape.

<p>For legend details related with the representation of the free energy minima and the pathways connecting them, see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004010#pcbi-1004010-g003" target="_blank">Fig. 3C</a>. Heme <i>a₃</i> is represented in grey sticks whereas the grey spheres correspond to the Cu atom from the Cu_B center and to the Fe atom from heme <i>a₃</i>. The residues forming the O₂ channels are shown in sticks with a sequence label. <b>A</b>- Lowest free energy pathway and free energy minima for the O₂ channel 1. The free energy values of the represented minima (in kJ·mol⁻¹) are: −21.07 (M₁), −11.79 (M₂), −10.58 (M₃), −17.97 (M₄), −17.53 (M₅), −16.25 (M₆), −13.87 (M₇), −20.07 (M₈), −20.47 (M₉), −20.47 (M₁₀) and −7.12 (M₁₁). <b>B</b>- Lowest free energy pathways and minima for the O₂ channel 2 and channel 3. For the O₂ channel 2, the values (in kJ·mol⁻¹) for the minima are: −13.87 (M₁₂), 0.14 (M₁₃), −22.77 (M₁₉) and −11.03 (M₂₀). The free energy values (in kJ·mol⁻¹) of the minima forming the O₂ channel 3 are: −17.24 (M₁₄), −10.46 (M₁₅), −15.81 (M₁₆), −10.40 (M₁₇), −10.18 (M₁₈), −22.77 (M₁₉) and −11.03 (M₂₀).</p