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Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer.

Andrews FH, Rogers MP, Paul LN, McLeish MJ - Biochemistry (2014)

Bottom Line: Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity.It was also found to be catalytically inactive.Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana 46202, United States.

ABSTRACT
The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

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A306F variant provided an unexpected rearrangementin the A–Dinterface. (A) wt BFDC showing the original positionsof Met145 and Arg141. (B) Phe306 causes Met145 and Arg141 to adoptnew rotamers, the latter stabilized by interaction with Glu134. Bluedashes indicate a distance of ∼3 Å. Red dashes indicatedistances of ≤4 Å.
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fig8: A306F variant provided an unexpected rearrangementin the A–Dinterface. (A) wt BFDC showing the original positionsof Met145 and Arg141. (B) Phe306 causes Met145 and Arg141 to adoptnew rotamers, the latter stabilized by interaction with Glu134. Bluedashes indicate a distance of ∼3 Å. Red dashes indicatedistances of ≤4 Å.

Mentions: Perhaps the most surprising observationwas that the A306F substitutionresulted in the displacement of the side chain of Arg141. This meantthat the guanidinium group of arginine forms an intramonomer saltbridge with the side chain of Glu134 rather than the intermonomerinteraction with Glu107 (Figure 8). The netresult was that the additional bulk of the new phenylalanine residuewas readily accommodated.


Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer.

Andrews FH, Rogers MP, Paul LN, McLeish MJ - Biochemistry (2014)

A306F variant provided an unexpected rearrangementin the A–Dinterface. (A) wt BFDC showing the original positionsof Met145 and Arg141. (B) Phe306 causes Met145 and Arg141 to adoptnew rotamers, the latter stabilized by interaction with Glu134. Bluedashes indicate a distance of ∼3 Å. Red dashes indicatedistances of ≤4 Å.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215898&req=5

fig8: A306F variant provided an unexpected rearrangementin the A–Dinterface. (A) wt BFDC showing the original positionsof Met145 and Arg141. (B) Phe306 causes Met145 and Arg141 to adoptnew rotamers, the latter stabilized by interaction with Glu134. Bluedashes indicate a distance of ∼3 Å. Red dashes indicatedistances of ≤4 Å.
Mentions: Perhaps the most surprising observationwas that the A306F substitutionresulted in the displacement of the side chain of Arg141. This meantthat the guanidinium group of arginine forms an intramonomer saltbridge with the side chain of Glu134 rather than the intermonomerinteraction with Glu107 (Figure 8). The netresult was that the additional bulk of the new phenylalanine residuewas readily accommodated.

Bottom Line: Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity.It was also found to be catalytically inactive.Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana 46202, United States.

ABSTRACT
The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

Show MeSH