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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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ThDP(yellow) in the active site of BFDC. The cofactor interactswith both the phosphate-binding domain of monomer A (green) and thepyrimidine-binding domain of monomer B (orange). Prepared using PyMOLusing data from PDB entry 1BFD.
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fig1: ThDP(yellow) in the active site of BFDC. The cofactor interactswith both the phosphate-binding domain of monomer A (green) and thepyrimidine-binding domain of monomer B (orange). Prepared using PyMOLusing data from PDB entry 1BFD.

Mentions: Such is the case with thiamin diphosphate (ThDP)-dependentenzymes.12,13 While this group of enzymes has evolvedto catalyze a wide rangeof chemical reactions,14 X-ray structureshave shown that overwhelmingly they have developed only one way tobind the ThDP cofactor, namely at the interface between two monomers.This creates two active sites per homodimer with each monomer contributingeither a pyrimidine binding (PYR) domain or a diphosphate binding(PP) domain to a given active site (Figure 1). At a minimum, therefore, any ThDP-dependent enzyme must form adimer.12,13 Curiously, until recently, all the ThDP-dependentenzymes whose sole in vivo function is to catalyzea decarboxylation reaction were found to be homotetramers, albeitmore accurately described as dimers of active dimers.15−21 The branched-chain 2-keto acid decarboxylase from Lactococcuslactis (KdcA, EC 4.1.1.72) was the first to deviate fromthis trend when its X-ray structure [Protein Data Bank (PDB) entry 2VBG] showed to it bedimeric.22 Intriguingly, although the archetypalThDP-dependent decarboxylase, pyruvate decarboxylase from Saccharomyces cerevisiae (ScPDC, EC 4.1.1.1),is a tetramer in the crystal lattice, analytical ultracentrifugation(AUC) experiments showed it to be a dimer at low solution concentrations.23 Further, using urea as a denaturant, it waspossible to shift the dimer–tetramer equilibrium in favor ofthe dimer and to demonstrate that the dimer was catalytically active.24,25


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

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

ThDP(yellow) in the active site of BFDC. The cofactor interactswith both the phosphate-binding domain of monomer A (green) and thepyrimidine-binding domain of monomer B (orange). Prepared using PyMOLusing data from PDB entry 1BFD.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: ThDP(yellow) in the active site of BFDC. The cofactor interactswith both the phosphate-binding domain of monomer A (green) and thepyrimidine-binding domain of monomer B (orange). Prepared using PyMOLusing data from PDB entry 1BFD.
Mentions: Such is the case with thiamin diphosphate (ThDP)-dependentenzymes.12,13 While this group of enzymes has evolvedto catalyze a wide rangeof chemical reactions,14 X-ray structureshave shown that overwhelmingly they have developed only one way tobind the ThDP cofactor, namely at the interface between two monomers.This creates two active sites per homodimer with each monomer contributingeither a pyrimidine binding (PYR) domain or a diphosphate binding(PP) domain to a given active site (Figure 1). At a minimum, therefore, any ThDP-dependent enzyme must form adimer.12,13 Curiously, until recently, all the ThDP-dependentenzymes whose sole in vivo function is to catalyzea decarboxylation reaction were found to be homotetramers, albeitmore accurately described as dimers of active dimers.15−21 The branched-chain 2-keto acid decarboxylase from Lactococcuslactis (KdcA, EC 4.1.1.72) was the first to deviate fromthis trend when its X-ray structure [Protein Data Bank (PDB) entry 2VBG] showed to it bedimeric.22 Intriguingly, although the archetypalThDP-dependent decarboxylase, pyruvate decarboxylase from Saccharomyces cerevisiae (ScPDC, EC 4.1.1.1),is a tetramer in the crystal lattice, analytical ultracentrifugation(AUC) experiments showed it to be a dimer at low solution concentrations.23 Further, using urea as a denaturant, it waspossible to shift the dimer–tetramer equilibrium in favor ofthe dimer and to demonstrate that the dimer was catalytically active.24,25

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