Multiple structure-based sequence alignment of FeADHs with a known 3D structure (residues 1–250 according to human ADHFE1).

<p>These proteins belong to five different subfamilies of the FeADH family. For comparison, ADHFE1 sequence from human is included in the alignment, as well as four glycerol dehydrogenase sequences with a known three-dimensional structure. PDB accession number of each sequence is indicated at the left side of alignment, whereas the protein subfamily to which each sequence belongs, is in the right side of the alignment. Conserved β-strands and α-helices for each structure are indicated in yellow and green, respectively. Residue position determinant for coenzyme specificity is indicated with a red square. Residues involved in the binding of Fe atom are highlighted in pink; residues involved in the binding of Zinc atom in glicerol dehydrogenases are highlighted in grey. Amino acid residues from human ADHFE1 sequence, highlighted in blue and grey indicate positions that belong to the N-terminal or C-terminal domains, respectively. The three-dimensional alignment of FeADH structures was performed using the VAST tool at the NCBI’s server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166851#pone.0166851.ref043" target="_blank">43</a>].</p

Phylogenetic analysis of 867 Fe-ADH protein sequences from eukaryotes plus 352 non-redundant sequences retrieved from the NCBI’s Conserved Domain Database (CDD).

<p>The evolutionary history was inferred using the Maximum Likelihood method based on the Le-Gascuel model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166851#pone.0166851.ref001" target="_blank">1</a>]. The tree with the highest log likelihood (-3414819.0869) is shown. Initial tree(s) for the heuristic search was/were obtained automatically applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4901)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 1219 amino acid sequences. There were a total of 996 positions in the final dataset.</p

Sequence logos of selected positions in different FeADH subfamilies.

<p>The sequences of FeADH family were sorted in subfamilies according to the results of the phylogenetic analysis. Numbering is according to the sequence of human ADHFE1. Residue in position 81 is determinant for coenzyme preference; residues in positions 242, 246, 330 and 357 are involved in metal binding. The amino acid residue coloring scheme was according to their chemical properties: polar (G, S, T, Y, C), green; neutral (Q, N), purple; basic (K, R, H), blue; acidic (D, E), red; and hydrophobic (A, V, L, I, P, M, W, F), black. FeADH subfamilies, whose members putatively use NADP⁺ as coenzyme, are enclosed with a blue box, those that use NAD⁺ as coenzyme, are enclosed with a red box, and those that use both NAD⁺ and NADP⁺, are enclosed in a green box. FeADH subfamilies with experimental support for coenzyme preference are indicated with an asterisk. Sequence logos were made using WebLogo 3 (<a href="http://weblogo.threeplusone.com" target="_blank">http://weblogo.threeplusone.com</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166851#pone.0166851.ref051" target="_blank">51</a>].</p

Diversity and Evolutionary Analysis of Iron-Containing (Type-III) Alcohol Dehydrogenases in Eukaryotes

<div><p>Background</p><p>Alcohol dehydrogenase (ADH) activity is widely distributed in the three domains of life. Currently, there are three non-homologous NAD(P)⁺-dependent ADH families reported: Type I ADH comprises Zn-dependent ADHs; type II ADH comprises short-chain ADHs described first in Drosophila; and, type III ADH comprises iron-containing ADHs (FeADHs). These three families arose independently throughout evolution and possess different structures and mechanisms of reaction. While types I and II ADHs have been extensively studied, analyses about the evolution and diversity of (type III) FeADHs have not been published yet. Therefore in this work, a phylogenetic analysis of FeADHs was performed to get insights into the evolution of this protein family, as well as explore the diversity of FeADHs in eukaryotes.</p><p>Principal Findings</p><p>Results showed that FeADHs from eukaryotes are distributed in thirteen protein subfamilies, eight of them possessing protein sequences distributed in the three domains of life. Interestingly, none of these protein subfamilies possess protein sequences found simultaneously in animals, plants and fungi. Many FeADHs are activated by or contain Fe²⁺, but many others bind to a variety of metals, or even lack of metal cofactor. Animal FeADHs are found in just one protein subfamily, the hydroxyacid-oxoacid transhydrogenase (HOT) subfamily, which includes protein sequences widely distributed in fungi, but not in plants), and in several taxa from lower eukaryotes, bacteria and archaea. Fungi FeADHs are found mainly in two subfamilies: HOT and maleylacetate reductase (MAR), but some can be found also in other three different protein subfamilies. Plant FeADHs are found only in chlorophyta but not in higher plants, and are distributed in three different protein subfamilies.</p><p>Conclusions/Significance</p><p>FeADHs are a diverse and ancient protein family that shares a common 3D scaffold with a patchy distribution in eukaryotes. The majority of sequenced FeADHs from eukaryotes are distributed in just two subfamilies, HOT and MAR (found mainly in animals and fungi). These two subfamilies comprise almost 85% of all sequenced FeADHs in eukaryotes.</p></div

Multiple structure-based sequence alignment of FeADHs with a known 3D structure (residues 251–467 according to human ADHFE1).

<p>For additional details see caption of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166851#pone.0166851.g005" target="_blank">Fig 5</a>.</p

Protein subfamilies that comprise the FeADH family.

<p>Protein subfamilies that comprise the FeADH family.</p

Unrooted tree constructed with protein sequences that possess homology to iron-dependent ADHs.

<p>2459 nonredundant protein sequences were retrieved from Protein Data Bank, Swiss Prot database, NCBI’s Conserved Domain Database, and Pfam database (using RP15 option to allow maximum representation of divergent proteins). Amino acid sequences were ascribed to protein families as considered by Pfam database (A) or NCBI’s Conserved Domain Database (B).</p

Number of FeADH proteins from plants found in different subfamilies.

<p>Number of FeADH proteins from plants found in different subfamilies.</p

Phylogenetic analysis of 538 Fe-ADH protein sequences retrieved from the NCBI’s Conserved Domain Database (CDD).

<p>The unrooted phylogenetic tree was inferred using the Maximum Likelihood method based on the Le-Gascuel model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166851#pone.0166851.ref049" target="_blank">49</a>]. Branches are colored according to the Conserved Domain Database Fe-ADH subfamily they belong. The tree with the highest log likelihood (-2505413,5328) is shown. Similar trees were obtained with maximum-parsimony, minimum-evolution and neighbour-joining methods. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.8682)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There were a total of 783 positions in the final dataset. The proportion of replicate trees in which the associated taxa clustered together in a bootstrap test (500 replicates) is given in color next to selected branches. Rectangles and triangles adjacent to each Fe-ADH subfamily name, indicate the presence of protein sequences from archaea domain (triangles), or eukarya domain (rectangles with A (animals), F (fungi), V (viridiplantae), and P (other eukaryotes) in each subfamily. Protein sequences from bacteria are present in all FeADH subfamilies.</p

New Insights on the Mechanism of the K⁺-Independent Activity of Crenarchaeota Pyruvate Kinases

<div><p>Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K⁺-dependent, whereas lysine confers K⁺-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K⁺-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K⁺ or an internal positive charge, the <i>Thermofilum pendens</i> pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K⁺-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K⁺ or the mutant E117K in the absence of K⁺. Thus, the substrate binding order of the <i>T</i>. <i>pendens</i> enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (<i>T_m</i> of 99.2°C), and the other (<i>T_m</i> of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (<i>T_m</i> of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in <i>Pyrobaculum aerophilum</i> and modeled in <i>T</i>. <i>pendens</i> were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the <i>T</i>. <i>pendens</i> pyruvate kinase could allow charge-independent catalysis.</p></div