4MQ5
Crystal Structure of Benzoylformate Decarboxylase Mutant A306F
Summary for 4MQ5
| Entry DOI | 10.2210/pdb4mq5/pdb |
| Related | 4MPP 4MPR |
| Descriptor | Benzoylformate decarboxylase, CALCIUM ION, THIAMINE DIPHOSPHATE, ... (5 entities in total) |
| Functional Keywords | thdp-dependent, cytol, lyase |
| Biological source | Pseudomonas putida |
| Total number of polymer chains | 1 |
| Total formula weight | 58143.54 |
| Authors | Andrews, F.H.,McLeish, M.J. (deposition date: 2013-09-15, release date: 2014-08-27, Last modification date: 2024-02-28) |
| Primary citation | Andrews, F.H.,Rogers, M.P.,Paul, L.N.,McLeish, M.J. Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer. Biochemistry, 53:4358-4367, 2014 Cited by PubMed 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. PubMed: 24956165DOI: 10.1021/bi500081r PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.502 Å) |
Structure validation
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