Additional file 1: Table S1. of Computational studies of human class V alcohol dehydrogenase - the odd sibling

Nomenclature for Human Alcohol Dehydrogenase. (PDF 36 kb

Sequence conservation characteristics of the individual RBDs.

<p>A. Consensus sequences of RBDs 1–6. The conserved residues are ordered according to frequency, with the most frequently occurring amino acid residue at the top. Rbm19 (human) residues are shown in bold. Secondary structure elements are derived as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043786#pone-0043786-g001" target="_blank">Fig. 1B</a>. B. Extent of conservation along each RBD. A window of five residues was slided along each of the six consensus RBDs, calculating the average presence of conserved residues (0–1, y-axis). This value is assigned to the central position of the window (solid line). The positions of α-helices are indicated in green and β-strands in red. C. Conserved residues in the 3D-structures of RBD2–6. Ribbon diagrams showing the 3D-structures of the human RBD2 (PDB identifier 2DGW) and the mouse RBD3–6 (PDB identifiers 1WHW, 1WHX, 2CPF and 2CPH, respectively), all in the same orientation, facing the β-sheet and with loops 1, 3 and 5 pointing downwards. Conserved residues are shown in red. In each RBD, the α1- and α2-helices as well as the β-strands (β1–β4) are labelled.</p

Dendrogram showing relationships between the RBDs.

<p>The six different RBDs form six clearly separated clusters, of which RBDs 1, 5 and 6 are most easily discernible. Each RBD is denoted with a two-letter code for species and a digit for the RBD number. Sequences are taken from: hs – <i>Homo sapiens</i> (Q9Y4C8), da – <i>Drosophila ananassae</i> (B3MYP1), ss – <i>Salpingoeca sp</i> (F2U536), mb – <i>Monosiga brevicollis</i> (A9USE7), cb – <i>Caenorhabditis briggsae</i> (A8WV73), bd – <i>Batrachochytrium dendrobatidis</i> (F4NSW1), dd – <i>Dictyostelium discoideum</i> (Q54PB2), tp – <i>Thalassiosira pseudonana</i> (B8BZC4), pi – <i>Phytophthora infestans</i> (D0NJ71), es – <i>Ectocarpus siliculosus</i> (D8LH81), at – <i>Arabidopsis thaliana</i> (F4JT92), ol – <i>Ostreococcus lucimarinus</i> (A4RVV1), tg – <i>Toxoplasma gondii</i> (B6KPW8), pm – <i>Perkinsus marinus</i> (C5KH14), sc – <i>Saccharomyces cerevisiae</i> (Q06106), pe – <i>Paramecium tetraurelia</i> (A0DWV5), ed – <i>Entamoeba dispar</i> (B0ECZ6), tc – <i>Trypanosoma cruzi</i> (E7KXH4), ng – <i>Naegleria gruberi</i> (D2V9G7), co – <i>Capsaspora owczarzaki</i> (E9C5E6), gl – <i>Giardia intestinalis</i> (A8BKE6). The number after each abbreviation indicates the RBD position.</p

Alignment of the microsporidia Rbm19/Mrd1 homologues to <i>S. cerevisiae</i> Mrd1.

<p>Alignment of RBDs 1, 3, 4, 6 and linker 3 of <i>S. cerevisiae</i> Mrd1 (denoted y) to <i>E. bieneusi</i> Mrd1 (denoted e). Identical residues (dark grey) or similar (light grey) between the two homologues are indicated. Secondary structure predictions are shown above the sequences. Positions present in the general consensus sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043786#pone-0043786-g005" target="_blank">Fig. 5A</a>) are underlined and asterisks indicate where 2 out of 3 of the microsporidia homologues (B7XJ60, C4V7E1 and E0S816) are conserved. Q8SRD9 was excluded due to high sequence similarity to E0S816 (>80% in the RBDs), in order to avoid bias.</p

Phylogenetic tree of eukaryotic lineages showing Rbm19/Mrd1 proteins containing five or six RBDs.

<p>The tree is redrawn from <a href="http://tolweb.og/Eukaryotes/" target="_blank">http://tolweb.og/Eukaryotes/</a>. Blue lines indicate organisms with five RBDs and green lines those with six RBDs. Grey lines indicate branches for which no completely sequenced genome is known yet. Dashed lines indicate uncertainties in the tree topology. Microsporidia are fungi, but exceptionally have only four RBDs.</p

Functionally Important Amino Acids in the <i>Arabidopsis</i> Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis

The anion transporter 1 (ANTR1) from <i>Arabidopsis thaliana</i>, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in <i>Escherichia coli</i>, ANTR1 mediates a Na⁺-dependent active transport of inorganic phosphate (P_i). The aim of this study was to identify amino acid residues involved in P_i binding and translocation by ANTR1 and in the Na⁺ dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from <i>E. coli</i> as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in <i>E. coli</i> and radioactive P_i transport assays and compared with bacterial cells carrying an empty plasmid. The results from P_i- and Na⁺-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na⁺ ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters

Mutational analysis of loop 5-β4 in RBD6 of Mrd1.

<p>A. The amino acid sequence of loop 5-β4 is shown for RBD6 and RBD5 of Mrd1 in comparison with the corresponding consensus sequences and secondary structure predictions. The analysed amino acid residues are shown in red. B. Growth characteristics of mutant <i>S. cerevisiae</i> cells compared to wild type. A dilution series (from left to right) of each mutant cell was spotted onto a selective FAA agar plate. The relevant amino acid sequence for each strain is shown to the right. Residues in red indicate the tested amino acid substitutions. The last mutant strain has the RBD6 sequence exchanged for the corresponding RBD5 sequence. C. Northern blot analysis of rRNA and pre-rRNA in the two mutant cells with impaired growth. The wild type <i>MRD1</i> gene under the control of a GAL promoter was shut off by growth in glucose medium for the indicated number of hours. Top panel shows the levels of 25S and 18S rRNA are shown after methylene blue staining of the membrane. Bottom panel shows the levels of the 35S, 23S and 20S pre-rRNAs are shown after hybridization with an oligonucleotide probe.</p

Properties of the linker regions.

<p>A. Disorder prediction of Rbm19 (human) and Mrd1 (<i>S. cerevisiae</i>). Grey areas indicate the RBDs: the red line represents the 0.05 threshold above which values are considered to indicate disorder. RBDs (RBD1–6) and linkers (L1–5) are indicated. B. Consensus sequence of linker 3. Conserved residues are ordered according to frequency, with the most common residue at the top. Rbm19 (human) residues are in bold. The secondary structure prediction given above the sequence (H = α-helix) is for linker 3 in Rbm19 (human).</p

Functionally Important Amino Acids in the <i>Arabidopsis</i> Thylakoid Phosphate Transporter: Homology Modeling and Site-Directed Mutagenesis

The anion transporter 1 (ANTR1) from <i>Arabidopsis thaliana</i>, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in <i>Escherichia coli</i>, ANTR1 mediates a Na⁺-dependent active transport of inorganic phosphate (P_i). The aim of this study was to identify amino acid residues involved in P_i binding and translocation by ANTR1 and in the Na⁺ dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from <i>E. coli</i> as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in <i>E. coli</i> and radioactive P_i transport assays and compared with bacterial cells carrying an empty plasmid. The results from P_i- and Na⁺-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na⁺ ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters

Genomic origin of bacterial contigs.

<p>The genomic origin of bacterial contigs as well as derived reads, DNA and RNA reads) based on closest homologs.</p