scholarly journals Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

2019 ◽  
Vol 8 (1) ◽  
pp. 40 ◽  
Author(s):  
Beatriz Hernández-Ochoa ◽  
Saúl Gómez-Manzo ◽  
Erick Alcaraz-Carmona ◽  
Hugo Serrano-Posada ◽  
Sara Centeno-Leija ◽  
...  
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Recombinant Expression ◽  
Glycolytic Enzyme ◽  
Kinetic Characteristics ◽  
Structural Determinants ◽  
Future Studies ◽  
Optimal Activity ◽  
Expression Characterization ◽  
Potential Use

Triosephosphate isomerase (TPI) is a glycolysis enzyme, which catalyzes the reversible isomerization between dihydroxyactetone-3-phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). In pathogenic organisms, TPI is essential to obtain the energy used to survive and infect. Fusarium oxisporum (Fox) is a fungus of biotechnological importance due to its pathogenicity in different organisms, that is why the relevance of also biochemically analyzing its TPI, being the first report of its kind in a Fusarium. Moreover, the kinetic characteristics or structural determinants related to its function remain unknown. Here, the Tpi gene from F. oxysporum was isolated, cloned, and overexpressed. The recombinant protein named FoxTPI was purified (97% purity) showing a molecular mass of 27 kDa, with optimal activity at pH 8.0 and and temperature of 37 °C. The values obtained for Km and Vmax using the substrate GAP were 0.47 ± 0.1 mM, and 5331 μmol min−1 mg−1, respectively. Furthemore, a protein structural modeling showed that FoxTPI has the classical topology of TPIs conserved in other organisms, including the catalytic residues conserved in the active site (Lys12, His94 and Glu164). Finally, when FoxTPI was analyzed with inhibitors, it was found that one of them inhibits its activity, which gives us the perspective of future studies and its potential use against this pathogen.

The kinetic consequences of altering the catalytic residues of triosephosphate isomerase

1986 ◽  
Vol 317 (1540) ◽  
pp. 371-380 ◽  
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Glycolytic Enzyme ◽  
Enzyme Mechanism ◽  
Site Directed Mutagenesis ◽  
Dihydroxyacetone Phosphate ◽  
Free Energies ◽  
Wild Type ◽  
Basic Residue ◽  
And Function

The essential basic residue at the active site of the glycolytic enzyme triosephosphate isomerase is Glu-165, which is responsible for the abstraction of either the 1-pro-R proton of dihydroxyacetone phosphate or of the 2-proton of D-glyceraldehyde 3-phosphate, in the enolization steps that constitute the reaction catalysed by this enzyme. We have changed this residue to Asp by oligonucleotide-mediated site-directed mutagenesis, and have evaluated the free-energy profile for the mutant protein. Comparison of the detailed energetics of the wild-type and mutant enzymes shows that only the transition-state free energies have been seriously affected, each of the enolization steps having been slowed by a factor of about one thousand. Evidently the movement of a catalytic group by less than 1 Å (1 Å = 10 -1 nm = 10 -10 m) has dramatic effects on catalysis, and the nature of these effects can provide important information about enzyme mechanism and function.

scholarly journals Conformational dynamics of active site loops 5, 6 and 7 of enzyme Triosephosphate Isomerase: A molecular dynamics study

2018 ◽  
Author(s):  
Sarath Chandra Dantu ◽  
Gerrit Groenhof
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Glycolytic Enzyme ◽  
Dihedral Angles ◽  
Dihydroxyacetone Phosphate ◽  
Closed Conformation ◽  
N Terminus

AbstractTriosephosphate Isomerase is a glycolytic enzyme catalyzing the interconversion of Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate. The active site is comprised of three distinct loops loop-6, loop-7 and loop-8. Based on loop-6 and loop-7 conformation we describe the enzyme as Open TIM and Closed TIM. Various NMR, X-ray crystallography and QM/MM simulation techniques have provided glimpses of individual events of what is essentially a dynamic process. We studied the conformational changes of two distinct loops (loop-6 and loop-7) enveloping the active site, in the presence of natural substrate, reaction intermediates and inhibitor molecules, by means of microsecond atomistic MD simulations in solution and crystal environment. Our studies have revealed that loop-6 samples open and closed conformations in both apo and holo TIM structures. As seen in solution state NMR experiments, we also observe that loop-6 N-terminus and C-terminus move independently. In our simulations we have also observed that backbone dihedrals of loop-7 residues G210 (G210-phi, G210-psi) and G211 (G211-phi) sample open and closed states in both apo and holo TIM structures. Whereas backbone dihedral angles of G211 (G211-psi) and S212 (S212-phi) adopt closed conformation only when the ligand is bound to the active site. As observed in chain-B of 1R2R crystal structures, we also observe that water molecules can also initiate flip of G211-psi and S212-phi dihedral angles into closed conformation. Except, loop-5, which has a dominant effect on the conformational behaviour of loop-6 N-terminus, we do not observe any influence of either loop-6 or loop-7 on the conformational dynamics of the other.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

1990 ◽  
Vol 171 (2) ◽  
pp. 565-570 ◽  
Author(s):  
K Ritter ◽  
H Brestrich ◽  
B Nellen ◽  
H Kratzin ◽  
H Eiffert ◽  
...  
Keyword(s):  
Infectious Mononucleosis ◽  
Triosephosphate Isomerase ◽  
Clinical Symptoms ◽  
Glycolytic Enzyme ◽  
Amino Acid Sequences ◽  
Cellular Proteins ◽  
51Cr Release ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Terminal Amino

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

1991 ◽  
Vol 10 (1) ◽  
pp. 50-69 ◽  
Author(s):  
Martin E. M. Noble ◽  
Rik K. Wierenga ◽  
Anne-Marie Lambeir ◽  
Fred R. Opperdoes ◽  
Andy-Mark W. H. Thunnissen ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Triosephosphate Isomerase

scholarly journals In silicoidentification of putativeTrypanosoma cruzienolase inhibitors

2019 ◽  
Author(s):  
Edward A. Valera-Vera ◽  
Melisa Sayé ◽  
Chantal Reigada ◽  
Mariana R. Miranda ◽  
Claudio A. Pereira
Keyword(s):  
Molecular Dynamics ◽  
Molecular Docking ◽  
Virtual Screening ◽  
Active Site ◽  
Screening Strategy ◽  
Glycolytic Enzyme ◽  
Docking Simulations ◽  
Enzyme Active Site ◽  
Key Enzyme

AbstractEnolase is a glycolytic enzyme that catalyzes the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In trypanosomatids enolase was proposed as a key enzyme afterin silicoandin vivoanalysis and it was validated as a protein essential for the survival of the parasite. Therefore, enolase constitutes an interesting enzyme target for the identification of drugs against Chagas disease. In this work, a combined virtual screening strategy was implemented, employing similarity virtual screening, molecular docking and molecular dynamics. First, two known enolase inhibitors and the enzyme substrates were used as queries for the similarity screening on the Sweetlead database using five different algorithms. Compounds retrieved in the top 10 of at least three search algorithms were selected for further analysis, resulting in six compounds of medical use (etidronate, pamidronate, fosfomycin, acetohydroximate, triclofos, and aminohydroxybutyrate). Molecular docking simulations predicted acetohydroxamate and triclofos would not bind to the active site of the enzyme, and a re-scoring of the obtained poses signaled fosfomycin and aminohydroxybutyrate as bad enzyme binders. Docking poses obtained for etidronate, pamidronate, and PEP, were used for molecular dynamics calculations to describe their mode of binding. From the obtained results, we propose etidronate as a possibleTcENO inhibitor, and describe desirable and undesirable molecular motifs to be taken into account in the repurposing or design of drugs aiming this enzyme active site.

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

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