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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.

Show MeSH
Interactions at the A–Dinterface. (A) Tyr288 of monomerA is found to have 12 points of contact (≤5 Å) with monomerD. (B) Ala306 of monomer A is located in a shallow hydrophobic cleftof monomer D.
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fig3: Interactions at the A–Dinterface. (A) Tyr288 of monomerA is found to have 12 points of contact (≤5 Å) with monomerD. (B) Ala306 of monomer A is located in a shallow hydrophobic cleftof monomer D.

Mentions: The A/D interface was the next largest,with a total surface area of ∼1600 Å2 or about8% of the total monomer A surface area. Here Tyr288 and Ala306 wereidentified as possible candidates for mutagenesis. Tyr288 of monomerA has 12 points of contact within 5 Å of monomer D (Figure 3), and it was reasoned that elimination of mostof these interactions by removal of the phenyl ring should resultin a less stable A/D interface. Additionally, the methyl side chainof Ala306 on monomer A is located in a shallow hydrophobic cleft ofmonomer D (Figure 3), and it was thought thatreplacement by a bulkier residue such as phenylalanine would not betolerated, and may help disrupt the interface.


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

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

Interactions at the A–Dinterface. (A) Tyr288 of monomerA is found to have 12 points of contact (≤5 Å) with monomerD. (B) Ala306 of monomer A is located in a shallow hydrophobic cleftof monomer D.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Interactions at the A–Dinterface. (A) Tyr288 of monomerA is found to have 12 points of contact (≤5 Å) with monomerD. (B) Ala306 of monomer A is located in a shallow hydrophobic cleftof monomer D.
Mentions: The A/D interface was the next largest,with a total surface area of ∼1600 Å2 or about8% of the total monomer A surface area. Here Tyr288 and Ala306 wereidentified as possible candidates for mutagenesis. Tyr288 of monomerA has 12 points of contact within 5 Å of monomer D (Figure 3), and it was reasoned that elimination of mostof these interactions by removal of the phenyl ring should resultin a less stable A/D interface. Additionally, the methyl side chainof Ala306 on monomer A is located in a shallow hydrophobic cleft ofmonomer D (Figure 3), and it was thought thatreplacement by a bulkier residue such as phenylalanine would not betolerated, and may help disrupt the interface.

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