Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a key enzyme in homofermentative metabolism where ethanol is the major product. PDCs are thiamine pyrophos­phate- and Mg2+ ion-dependent enzymes that catalyse the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. As this enzyme class is rare in bacteria, current knowledge of bacterial PDCs is extremely limited. One approach to further the understanding of bacterial PDCs is to exploit the diversity provided by evolution. Ancestral sequence reconstruction (ASR) is a method of computational molecular evolution to infer extinct ancestral protein sequences, which can then be synthesized and experimentally characterized. Through ASR a novel PDC was generated, designated ANC27, that shares only 78% amino-acid sequence identity with its closest extant homologue (Komagataeibacter medellinensis PDC, GenBank accession No. WP_014105323.1), yet is fully functional. Crystals of this PDC diffracted to 3.5 Å resolution. The data were merged in space group P3221, with unit-cell parameters a = b = 108.33, c = 322.65 Å, and contained two dimers (two tetramer halves) in the asymmetric unit. The structure was solved by molecular replacement using PDB entry 2wvg as a model, and the final R values were Rwork = 0.246 (0.3671 in the highest resolution bin) and Rfree = 0.319 (0.4482 in the highest resolution bin). Comparison with extant bacterial PDCs supports the previously observed correlation between decreased tetramer interface area (and number of interactions) and decreased thermostability

Evolutionary conserved motifs constrain the RNA structure organization of picornavirus IRES

Picornavirus RNAs initiate translation using a 5′ end-independent mechanism based on internal ribosome entry site (IRES) elements. Despite performing similar functions, IRES elements present in genetically distant RNAs differ in primary sequence, RNA secondary structure and trans-acting factors requirement. The lack of conserved features amongst IRESs represents obstacles for the understanding of the internal initiation process. RNA structure is tightly linked to picornavirus IRES activity, consistent with the conservation of RNA motifs. This study extends the functional relevance of evolutionary conserved motifs of foot-and-mouth disease virus (FMDV) IRES. SHAPE structural analysis of mutant IRESs revealed local changes in RNA flexibility indicating the existence of an interactive structure constrained by lateral bulges that maintain the RNA conformation necessary for IRES-mediated translation. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.BFU2011-25437; Fundación Ramón ArecesPeer Reviewe

Crystal structure of pyruvate decarboxylase from <i> Zymobacter palmae</i>

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- andMg2+ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions

Crystal structure of pyruvate decarboxylase from Zymobacter palmae

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg²⁺ ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rᵣ.ᵢ.ₘ. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwₒᵣₖ = 0.186 (0.271 in the highest resolution bin) and Rfᵣₑₑ = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions