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O2‐independent demethylation of trimethylamine N‐oxide by Tdm of Methylocella silvestris
- Zhu, Yijun, Ksibe, Amira Z., Schäfer, Hendrik, Blindauer, Claudia A., Bugg, Timothy D. H., Chen, Yin
- The FEBS journal 2016 v.283 no.21 pp. 3979-3993
- Methylocella, aspartic acid, catalytic activity, dimethylamine, formaldehyde, hydrogen bonding, hydrophobicity, iron, mass spectrometry, models, oxygen, site-directed mutagenesis, stoichiometry, trimethylamine, zinc
- Bacterial trimethylamine N‐oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen‐independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, site‐directed mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn²⁺ and 1 molar equivalent of non‐haem Fe²⁺. We further investigated Zn²⁺‐ and Fe²⁺‐binding sites through homology modelling and site‐directed mutagenesis and found that Zn²⁺ is coordinated by a 3‐sulfur‐1‐O motif. An aspartate residue (D198) likely bridges Fe²⁺ and Zn²⁺ centres, either directly or indirectly via H‐bonding through a neighbouring H₂O molecule. H276 contributes to Fe²⁺ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site‐directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation–π interaction. Furthermore, a crossover experiment using a substrate analogue gave direct evidence that a trimethylamine‐alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn²⁺ and Fe²⁺ in the catalysis of TMAO demethylation by this unique oxygen‐independent enzyme.