TIM-barrel Enzyme Research Articles

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.

Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid—the apicoplast—which originated from a secondary (eukaryote–eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(−) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.

A monomeric TIM-barrel structure fromPyrococcus furiosusis optimized for extreme temperatures

The structure of phosphoribosyl anthranilate isomerase (TrpF) from the hyperthermophilic archaeon Pyrococcus furiosus (PfTrpF) has been determined at 1.75 Å resolution. The PfTrpF structure has a monomeric TIM-barrel fold which differs from the dimeric structures of two other known thermophilic TrpF proteins. A comparison of the PfTrpF structure with the two known bacterial thermophilic TrpF structures and the structure of a related mesophilic protein from Escherichia coli (EcTrpF) is presented. The thermophilic TrpF structures contain a higher proportion of ion pairs and charged residues compared with the mesophilic EcTrpF. These residues contribute to the closure of the central barrel and the stabilization of the barrel and the surrounding α-helices. In the monomeric PfTrpF conserved structural water molecules are mostly absent; instead, the structural waters are replaced by direct side-chain-main-chain interactions. As a consequence of these combined mechanisms, the P. furiosus enzyme is a thermodynamically stable and entropically optimized monomeric TIM-barrel enzyme which defines a good framework for further protein engineering for industrial applications.

Molecular evolution of dihydrouridine synthases

BackgroundDihydrouridine (D) is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS). DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as “predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family”.ResultsTo elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model.ConclusionsWe compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS family provides a background to study functional differences among these proteins that will guide experimental analyses.

2P077 表面のアミノ酸組成を単純化した酵素の選択と解析(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

2P077 表面のアミノ酸組成を単純化した酵素の選択と解析(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling

A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T opt = 68 °C), and the hyperthermophilic xylanase B (TmxB, T opt = 102 °C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant ( T opt = 80 °C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N- and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N- and C-termini are in contact.

Crystal Structure of d-Psicose 3-epimerase from Agrobacterium tumefaciens and its Complex with True Substrate d-Fructose: A Pivotal Role of Metal in Catalysis, an Active Site for the Non-phosphorylated Substrate, and its Conformational Changes

d-Psicose, a rare sugar produced by the enzymatic reaction of d-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of d-fructose and d-psicose by epimerizing the C-3 position, with marked efficiency for d-psicose. The crystal structures of DPEase and its complex with the true substrate d-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of d-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the β4–α4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound d-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.

The TIM-barrel fold: a versatile framework for efficient enzymes

Recent studies on triosephosphate isomerase (TIM)-barrel enzymes highlight the remarkable versatility of the TIM-barrel scaffold. At least 15 distinct enzyme families use this framework to generate the appropriate active site geometry, always at the C-terminal end of the eight parallel β-strands of the barrel. Sequence and structure comparisons now suggest that many of the TIM-barrel enzymes are evolutionarily related. Common structural properties of TIM-barrel enzymes are discussed.

Stability of a thermophilic TIM-barrel enzyme: indole-3-glycerol phosphate synthase from the thermophilic archaeon Sulfolobus solfataricus.

The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature. In this paper we focus on three points: (1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature. Results can be summarized as follows: (a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0. We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0. Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14. 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0.

Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes

Indole-3-glycerol-phosphate synthase, a thermophilic and thermostable enzyme from the archaeon Sulfolobus solfataricus, was purified and characterized. The sequence of the thermophilic enzyme was compared to the sequence of a homologous mesophilic enzyme from Escherichia coli. The secondary structure of the thermophilic enzyme was predicted taking into account the patterns of hydropathy, chain flexibility and amphipathicity and the CD spectrum. From this analysis it turned out that indole-3-glycerol-phosphate synthase from S. solfataricus can be considered a model for studying thermostable TIM-barrel enzymes. Some peculiarities of the amino-acid sequence of indole-3-glycerol-phosphate synthase from S. solfataricus are discussed in relation to the thermostability of the enzyme.

Improving the prediction of secondary structure of ‘TIM-barrel’ enzymes

The information contained in aligned sets of homologous protein sequences should improve the score of secondary structure prediction. Seven different enzymes having the (beta/alpha)8 or TIM-barrel fold were used to optimize the prediction with regard to this class of enzymes. The alpha-helix, beta-strand and loop propensities of the Garnier-Osguthorpe-Robson method were averaged at aligned residue positions, leading to a significant improvement over the average score obtained from single sequences. The increased accuracy correlates with the average sequence variability of the aligned set. Further improvements were obtained by using the following averaged properties as weights for the averaged state propensities: amphipathic moment and alpha-helix; hydropathy and beta-strand; chain flexibility and loop. The clustering of conserved residues at the C-terminal ends of the beta-strands was used as an additional positive weight for beta-strand propensity and increased the prediction of otherwise unpredicted beta-strands decisively. The automatic weighted prediction method identifies greater than 95% of the secondary structure elements of the set of seven TIM-barrel enzymes.