Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case.

A coleopteran triosephosphate isomerase (TIM) from Tenebrio molitor (yellow mealworm beetle) was recombinantly expressed in Escherichia coli and characterized with respect to thermal stability, kinetic parameters and oligomeric state. The enzyme was successfully crystallized and the structure determined by X-ray analysis to 2.0 A resolution. This is the first example of an invertebrate TIM. We compare structural features with known structures of TIMs from microorganisms, plants and vertebrates, and discuss the utility of the Tenebrio TIM sequence, together with several newly sequenced insect TIMs, for molecular phylogenetic analysis.

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing six different TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. Interestingly, not all of the TPI alleles exhibit similar characteristics in terms of stability, temperature sensitivity and activity. Yeast with the M80T allele exhibits significant changes in growth and metabolic activity at all temperatures. Yeast with the E104D allele exhibit an acute temperature sensitive phenotype for growth and metabolic activity at 37C. All of the alleles except C41Y demonstrate protein instability at 37C. This study further examines the role of protein folding in TPI deficiency disease pathogenesis. Yeast expressing the mutant TPI alleles were examined for activation of HSF, a cytoplasmic protein folding stress response transcription factor. Understanding the activation of cell stress pathways in TPI deficient yeast may lead to understanding of disease pathogenesis in TPI deficiency patients and provide potential pharmacological targets.Support or Funding InformationResearch is supported by an SRU Student Faculty Research GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The fructose-1,6-bisphosphate aldolase catalyzed glycolysis branch that forms dihydroxyacetone phosphate and glyceraldehyde-3-phosphate was identified as a key driver of increased oil synthesis in oil palm and was validated in Saccharomyces cerevisiae. Reduction in triose phosphate isomerase (TPI) activity in a yeast knockdown mutant resulted in 19% increase in lipid content, while yeast strains overexpressing oil palm fructose-1,6-bisphosphate aldolase (EgFBA) and glycerol-3-phosphate dehydrogenase (EgG3PDH) showed increased lipid content by 16% and 21%, respectively. Genetic association analysis on oil palm SNPs of EgTPI SD_SNP_000035801 and EgGAPDH SD_SNP_000041011 showed that palms harboring homozygous GG in EgTPI and heterozygous AG in EgGAPDH exhibited higher mesocarp oil content based on dry weight. In addition, AG genotype of the SNP of EgG3PDH SD_SNP_000008411 was associated with higher mean mesocarp oil content, whereas GG genotype of the EgFBA SNP SD_SNP_000007765 was favourable. Additive effects were observed with a combination of favourable alleles in TPI and FBA in Nigerian x AVROS population (family F7) with highest allele frequency GG.GG being associated with a mean increase of 3.77% (p value = 2.3E−16) oil content over the Family 1. An analogous effect was observed in yeast, where overexpressed EgFBA in TPI- resulted in a 30% oil increment. These results provide insights into flux balances in glycolysis leading to higher yield in mesocarp oil-producing fruit.

The crystal structure of methylglyoxal synthase from Escherichia coli

The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90 degrees of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.

Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase

The enormous rate accelerations observedfor many enzyme catalystsare due to strong stabilizing interactions between the protein andreaction transition state. The defining property of these catalystsis their specificity for binding the transition state with a muchhigher affinity than substrate. Experimental results are presentedwhich show that the phosphodianion-binding energy of phosphate monoestersubstrates is used to drive conversion of their protein catalystsfrom flexible and entropically rich ground states to stiff and catalyticallyactive Michaelis complexes. These results are generalized to otherenzyme-catalyzed reactions. The existence of many enzymes in flexible,entropically rich, and inactive ground states provides a mechanismfor utilization of ligand-binding energy to mold these catalysts intostiff and active forms. This reduces the substrate-binding energyexpressed at the Michaelis complex, while enabling the full and specificexpression of large transition-state binding energies. Evidence ispresented that the complexity of enzyme conformational changes increaseswith increases in the enzymatic rate acceleration. The requirementthat a large fraction of the total substrate-binding energy be utilizedto drive conformational changes of floppy enzymes is proposed to favorthe selection and evolution of protein folds with multiple flexibleunstructured loops, such as the TIM-barrel fold. The effect of proteinmotions on the kinetic parameters for enzymes that undergo ligand-drivenconformational changes is considered. The results of computationalstudies to model the complex ligand-driven conformational change incatalysis by triosephosphate isomerase are presented.

Structure of glycolate oxidase from spinach at a resolution of 5.5 Å

The effect of selenium enrichment on baker's yeast proteome.

Triosephosphate isomerase has been shown to exist in two unliganded forms, one of which binds and isomerizes (R)-glyceraldehyde 3-phosphate and the other of which binds and isomerizes dihydroxyacetone 3-phosphate. The tracer perturbation method of Britton demonstrates the kinetic significance of the interconversion of these two enzyme forms at high substrate concentrations and yields a rate constant of about 10(6) s-1 for the interconversion. Although the molecular nature of the two forms of unliganded enzyme is not defined by these experiments, a shuffling of protons among active site residues, or a protein conformational change, or both, may be involved. This study, coupled with the known rate constants for the substrate-handling steps of triosephosphate isomerase catalysis, completes the kinetic characterization of the catalytic cycle for this enzyme.

Automatic and accurate method for analysis of proteins that undergo hinge-mediated domain and loop movements

In this work, we report the implications of the monomerization of triosephosphate isomerase (TIM) from T. cruzi (TcTIM). A monomeric mutant (monoTcTIM) of this species was constructed by genetic engineering, shortening the main loop of interdigitation, which is fundamental for its dimerization. The properties of monoTcTIM were compared with those of the other TIM monomeric versions: genetically engineered mutants, or equilibrium intermediates obtained by chemical denaturation. The stability for almost all the monomeric variants so far reported appears in the same range, and also presents similar structural characteristics. Regarding the catalytic activity of monomeric mutants, when it is present is in several orders of magnitude lower than those observed in their respective wild‐type enzyme. The change in the hydrophobic surface of TcTIM after and before monomerization was also studied and corresponds to an extensive area of the interface region, which becomes exposed when monomers are dissociated. This fact could be related with conformational changes in the local environment of the catalytic amino acids responsible for the isomerization of the substrate and consequently the inactivation of its catalytic properties. Comparison of the structural, folding and unfolding properties, as well as stability studies could give answers on why this enzyme is an obligate oligomer.

The glycolytic enzyme triosephosphate isomerase (TIM) catalyzes the interconversion of the three-carbon sugars dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) at a rate limited by the diffusion of substrate to the enzyme. We have solved the three-dimensional structure of TIM complexed with a reactive intermediate analogue, phosphoglycolohydroxamate (PGH), at 1.9-A resolution and have refined the structure to an R-factor of 18%. Analysis of the refined structure reveals the geometry of the active-site residues and the interactions they make with the inhibitor and, by analogy, the substrates. The structure is consistent with an acid-base mechanism in which the carboxylate of Glu-165 abstracts a proton from carbon while His-95 donates a proton to oxygen to form an enediol (or enediolate) intermediate. The conformation of the bound substrate stereoelectronically favors proton transfer from substrate carbon to the syn orbital of Glu-165. The crystal structure suggests that His-95 is neutral rather than cationic in the ground state and therefore would have to function as an imidazole acid instead of the usual imidazolium. Lys-12 is oriented so as to polarize the substrate oxygens by hydrogen bonding and/or electrostatic interaction, providing stabilization for the charged transition state. Asn-10 may play a similar role.

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

This research aims to analyze transient processes during loop closing and opening operations in distribution grids with DGs. The realization of load transfer without blackouts can improve the reliability of a power supply, reducing economic loss. Hence, we summarize the problems appearing during loop closing and opening processes. Based on this analysis, a loop closing operation was executed at one 110 kV substation, and the voltage and current waveform were recorded. A theoretical analysis of field experiments results yielded the spectral characteristics of the system. A loop closing operation was simulated using EMTP-ATP software. The simulation and the field experiment results matched closely, thus validating the simulation model. We conclude that closing and opening operation are influenced by voltage difference, including the amplitude and phase angle difference. The system capacitance and inductance have impact not only on the transient over- voltage, but also on the frequency characteristics of the system.