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Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

Bottom Line: In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer.Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model.The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

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In vivo characterization of TIMs complemented strains on glycerol minimal media in the presence of IPTG.(A) Complementation of point mutants into an E. coli DE3 ΔTIM strain. Transformed E. coli were grown in plates with glycerol as a carbon source in M63 minimal media and 0.1mM of IPTG. E. coli transformed with plasmids containing wild-type TvTIM1 are able to complement. Mutants I45A, I45V, I45F and I45L complement with similar efficiency as wild type, whereas mutants I45Y contained less colonies, and no colonies appear after 48 hrs for I45G and I445W. (B) Growth kinetics of E. coli complemented strains in liquid minimal media. Growth rates of cultures grown in minimal medium. Bacteria transformed with plasmids containing wild-type TvTIM1 and mutants I45L, I45V, complement with similar efficiency, whereas, mutants I45A, I45F, I45Y and I45G present slower growth rates and no growth was observed with the tryptophan mutant and the empty vector.
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pone.0141747.g003: In vivo characterization of TIMs complemented strains on glycerol minimal media in the presence of IPTG.(A) Complementation of point mutants into an E. coli DE3 ΔTIM strain. Transformed E. coli were grown in plates with glycerol as a carbon source in M63 minimal media and 0.1mM of IPTG. E. coli transformed with plasmids containing wild-type TvTIM1 are able to complement. Mutants I45A, I45V, I45F and I45L complement with similar efficiency as wild type, whereas mutants I45Y contained less colonies, and no colonies appear after 48 hrs for I45G and I445W. (B) Growth kinetics of E. coli complemented strains in liquid minimal media. Growth rates of cultures grown in minimal medium. Bacteria transformed with plasmids containing wild-type TvTIM1 and mutants I45L, I45V, complement with similar efficiency, whereas, mutants I45A, I45F, I45Y and I45G present slower growth rates and no growth was observed with the tryptophan mutant and the empty vector.

Mentions: Because of the decrease in catalytic efficiently observed in the monomeric mutants we were curious to assess the complementation of TvTIM mutants in vivo. An E. coli (DE3) ΔTIM strain [27] transformed with plasmids over expressing I45F, I45L, I45V, I45Y, and I45A mutants complemented in minimal media similarly than wild-type TvTIM1 after an incubation period of 48 h at 30°C. In contrast no colonies were present after a transformation with plasmids containing I45G and I45W mutants (Fig 3A). The growth rates of bacteria cultures in minimal medium indicates that bacteria transformed with plasmids containing wild-type and mutants I45L, I45V, I45A, I45F present similar growth rates, whereas I45Y and I45G present a slower growth rate. No growth was observed for I45W or an empty vector (Fig 3B).


Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

Lara-Gonzalez S, Estrella P, Portillo C, Cruces ME, Jimenez-Sandoval P, Fattori J, Migliorini-Figueira AC, Lopez-Hidalgo M, Diaz-Quezada C, Lopez-Castillo M, Trasviña-Arenas CH, Sanchez-Sandoval E, Gómez-Puyou A, Ortega-Lopez J, Arroyo R, Benítez-Cardoza CG, Brieba LG - PLoS ONE (2015)

In vivo characterization of TIMs complemented strains on glycerol minimal media in the presence of IPTG.(A) Complementation of point mutants into an E. coli DE3 ΔTIM strain. Transformed E. coli were grown in plates with glycerol as a carbon source in M63 minimal media and 0.1mM of IPTG. E. coli transformed with plasmids containing wild-type TvTIM1 are able to complement. Mutants I45A, I45V, I45F and I45L complement with similar efficiency as wild type, whereas mutants I45Y contained less colonies, and no colonies appear after 48 hrs for I45G and I445W. (B) Growth kinetics of E. coli complemented strains in liquid minimal media. Growth rates of cultures grown in minimal medium. Bacteria transformed with plasmids containing wild-type TvTIM1 and mutants I45L, I45V, complement with similar efficiency, whereas, mutants I45A, I45F, I45Y and I45G present slower growth rates and no growth was observed with the tryptophan mutant and the empty vector.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664265&req=5

pone.0141747.g003: In vivo characterization of TIMs complemented strains on glycerol minimal media in the presence of IPTG.(A) Complementation of point mutants into an E. coli DE3 ΔTIM strain. Transformed E. coli were grown in plates with glycerol as a carbon source in M63 minimal media and 0.1mM of IPTG. E. coli transformed with plasmids containing wild-type TvTIM1 are able to complement. Mutants I45A, I45V, I45F and I45L complement with similar efficiency as wild type, whereas mutants I45Y contained less colonies, and no colonies appear after 48 hrs for I45G and I445W. (B) Growth kinetics of E. coli complemented strains in liquid minimal media. Growth rates of cultures grown in minimal medium. Bacteria transformed with plasmids containing wild-type TvTIM1 and mutants I45L, I45V, complement with similar efficiency, whereas, mutants I45A, I45F, I45Y and I45G present slower growth rates and no growth was observed with the tryptophan mutant and the empty vector.
Mentions: Because of the decrease in catalytic efficiently observed in the monomeric mutants we were curious to assess the complementation of TvTIM mutants in vivo. An E. coli (DE3) ΔTIM strain [27] transformed with plasmids over expressing I45F, I45L, I45V, I45Y, and I45A mutants complemented in minimal media similarly than wild-type TvTIM1 after an incubation period of 48 h at 30°C. In contrast no colonies were present after a transformation with plasmids containing I45G and I45W mutants (Fig 3A). The growth rates of bacteria cultures in minimal medium indicates that bacteria transformed with plasmids containing wild-type and mutants I45L, I45V, I45A, I45F present similar growth rates, whereas I45Y and I45G present a slower growth rate. No growth was observed for I45W or an empty vector (Fig 3B).

Bottom Line: In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer.Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model.The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

View Article: PubMed Central - PubMed

Affiliation: IPICYT, División de Biología Molecular, Camino a la Presa San José 2055, CP 78216, San Luis Potosí, San Luis Potosí, México.

ABSTRACT
The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

Show MeSH
Related in: MedlinePlus