Limits...
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
Potential Glu141–Glu107interaction avoided in the R141Evariant. Instead, Glu141 is found coordinated to Trp125 and an additionalNa+ ion. Blue dashes indicate distances of ≤3 Å.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215898&req=5

fig7: Potential Glu141–Glu107interaction avoided in the R141Evariant. Instead, Glu141 is found coordinated to Trp125 and an additionalNa+ ion. Blue dashes indicate distances of ≤3 Å.

Mentions: It was predicted that mutating Arg141 to a glutamate would resultin a variant that not only would be unable to form a salt bridge withGlu107 but also would create like-charge repulsion between Glu107and Glu141. Surprisingly, the crystal structure of the R141E variantrevealed the carboxylate group of Glu141 is complexed to a Na+ ion, as well as the indole moiety of Trp125 (Figure 7). Four water molecules were also coordinated tothe sodium ion, as was the carbonyl of Gly105. One of these waterswas located within 3 Å of the carboxylate of Glu107. It seemsthat these unexpected interactions were sufficient to alleviate thepredicted charge–charge repulsion that was expected to tiltthe equilibrium toward the dimeric state.


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

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

Potential Glu141–Glu107interaction avoided in the R141Evariant. Instead, Glu141 is found coordinated to Trp125 and an additionalNa+ ion. Blue dashes indicate distances of ≤3 Å.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Potential Glu141–Glu107interaction avoided in the R141Evariant. Instead, Glu141 is found coordinated to Trp125 and an additionalNa+ ion. Blue dashes indicate distances of ≤3 Å.
Mentions: It was predicted that mutating Arg141 to a glutamate would resultin a variant that not only would be unable to form a salt bridge withGlu107 but also would create like-charge repulsion between Glu107and Glu141. Surprisingly, the crystal structure of the R141E variantrevealed the carboxylate group of Glu141 is complexed to a Na+ ion, as well as the indole moiety of Trp125 (Figure 7). Four water molecules were also coordinated tothe sodium ion, as was the carbonyl of Gly105. One of these waterswas located within 3 Å of the carboxylate of Glu107. It seemsthat these unexpected interactions were sufficient to alleviate thepredicted charge–charge repulsion that was expected to tiltthe equilibrium toward the dimeric state.

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