The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop

Borchert, Torben V.; Abagyan, Ruben; Kishan, KV Radha; Zeelen, J.P.; Wierenga, R.K.

doi:10.1016/0969-2126(93)90021-8

Cited by 75 publications

(58 citation statements)

References 22 publications

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“…Previously, we have shown that the 15 residues of loop-3 of trypanosomal TIM (wtTIM) can be replaced by an eight residue loop, in such a way that a stable, monomeric protein (monoTIM) is obtained [8]. The crystal structure of monoTIM has been reported [9]. Here we report on the characterisation of another interface variant of wtTIM: H47N.…”

Section: Introductionmentioning

confidence: 99%

An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

et al. 1995

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Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His-47 of wtTlM has been mutated into an asparagine. Ultracentrifagation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give Tm-values of 41°C for wtTIM, whereas the Tm-value for the monomeric form of H47N is approximately 7°C lower.

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Section: Introductionmentioning

confidence: 99%

An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

et al. 1995

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“…Although site-directed disruption of these enzymes resulted in complete loss of enzymatic activity, replacement of an interface loop of TIM resulted in a stable monomer with a 103-fold reduction in kcat (11,12). These studies have suggested that the quaternary structure is essential for proper catalytic activity.…”

mentioning

confidence: 98%

Disruption of the aldolase A tetramer into catalytically active monomers.

Beernink

Tolan

1996

Proc. Natl. Acad. Sci. U.S.A.

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The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Crosslinking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation.There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in. many proteins.

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“…As such this stable framework with tolerant sequence variations has already been used as an interesting scaffold to design loops (11,12) and even artificial proteins (13,14). The active sites of all these TIM barrel enzymes are located at the C-terminal end of the ␤-barrel, with catalytic residues contributed by the ␤-strands and the loops connecting the ␤-strands to subsequent ␣-helices numbered loop 1 to loop 8 in correspondence with the numbering of the preceding ␤-strands.…”

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confidence: 99%

Triose-phosphate Isomerase (TIM) of the Psychrophilic BacteriumVibrio marinus

Alvarez¹,

Zeelen²,

Mainfroid³

et al. 1998

Journal of Biological Chemistry

147

108

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The purification and characterization of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-Å resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15°C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25°C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The T d values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54°C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.Triose-phosphate isomerase (TIM 1 ; EC 5.3.1.1) is a dimeric glycolytic enzyme formed by two identical subunits consisting of about 250 residues each. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate (see Fig. 1). TIM has been the subject of extensive biophysical, enzymological, and computational studies. Its catalytic properties and mechanism have been studied in detail (1, 2). It has been established that TIM is a very efficient catalyst, since the reaction rates are diffusion-controlled. Neither cofactors nor metal ions are required in this reaction, and there is no evidence of allostery or cooperativity among the subunits.Structurally TIMs do form a well characterized family. To date, x-ray structures of TIM from seven different sources have been solved (wild type or/and in complex with substrate analogues): chicken (3), yeast (4), Trypanosoma brucei (5), Escherichia coli (6), human (7), Bacillus stearothermophilus (8), and Plasmodium falciparum (9). In addition, the structures have been determined of Leishmania mexicana TIM 2 and of Thermotoga maritima TIM. 3 Also, 45 amino acid TIM sequences from a wide variety of organisms have been determined and are available in the data bases.Each TIM monomer has a globular shape with two protruding loops. The globular part is formed by an 8-fold repeat of a ␤-strand, loop, ␣-helix, loop motif. The ␤␣ units fold up in a regular way, such that the ␤-strands (numbered ␤1 to ␤8) form an eight-stranded ␤-barrel surrounded by the eight ␣-helices (numbered ␣1 to ␣8) on the outside. This folding motif is also referred to as the TIM barrel motif. In TIM, the two protruding loops are after ␤-strand three and after ␤-strand six.The TIM barrel structural motif is found for a large number of other enzymes with widely different functions (10) that have little or no sequence homology. As such this stable framework with tolerant sequence variations has already been use...

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The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: the correct modelling of an eight-residue loop

Cited by 75 publications

References 22 publications

An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

An interface point‐mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

Disruption of the aldolase A tetramer into catalytically active monomers.

Triose-phosphate Isomerase (TIM) of the Psychrophilic BacteriumVibrio marinus

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