4MPR

Benzoylformate Decarboxylase: Is the tetramer vital for activity?

4MPR の概要

• エントリーDOI: 10.2210/pdb4mpr/pdb
• 関連するPDBエントリー: 4MPP 4MQS
• 分子名称: Benzoylformate decarboxylase, CALCIUM ION, THIAMINE DIPHOSPHATE, ... (7 entities in total)
• 機能のキーワード: thdp-dependent, cytol, lyase
• 由来する生物種: Pseudomonas putida
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 58328.11

構造登録者

Andrews, F.H.,McLeish, M.J. (登録日: 2013-09-13, 公開日: 2014-08-27, 最終更新日: 2024-02-28)

主引用文献

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

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: 24956165
DOI: 10.1021/bi500081r

実験手法

X-RAY DIFFRACTION (1.401 Å)