O₂‐independent demethylation of trimethylamine N‐oxide by Tdm of Methylocella silvestris

Abstract

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 Zn 2+ and 1 molar equivalent of non-haem Fe 2+ . We further investigated Zn 2+ -and Fe 2+ -binding sites through homology modelling and site-directed mutagenesis and found that Zn 2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe 2+ and Zn 2+ centres, either directly or indirectly via H-bonding through a neighbouring H 2 O molecule. H276 contributes to Fe 2+ 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-p 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 Zn 2+ and Fe 2+ in the catalysis of TMAO demethylation by this unique oxygen-independent enzyme. IntroductionBacterial trimethylamine N-oxide (TMAO) demethylase (Tdm) is a key enzyme involved in bacterial degradation of trimethylamine (TMA) and TMAO [1][2][3]. The enzyme was first proposed in the 1970s and has been partially purified from Bacillus sp. PM6 [4] and Pseudomonas aminovorans (now Aminobacter aminovorans [5]). Despite being purified from aerobic hosts, Tdm can convert TMAO anaerobically to equimolar amounts of dimethylamine (DMA) and formaldehyde (HCHO) (1 TMAO ? 1 DMA + 1 HCHO) [2,3,5]. The gene encoding Tdm has only been identified very recently and it is now known that tdm is widely Abbreviations ADH, alcohol dehydrogenase; Asc, ascorbic acid; BS-DMA, benzenesulfonyl dimethylamine adduct; BS-MEA, benzenesulfonyl methylethylamine adduct; DMEA, dimethyethylamine; EDTA, ethylenediaminetetraacetic acid; GCV_T, glycine cleavage T protein; HCHO, formaldehyde; HMS, hydroxymethanesulfonate; ICP, inductively coupled plasma-mass spectrometry; MS, mass spectrometer; OES, optical emission spectrometer; Tdm, trimethylamine N-oxide demethylase; THF, tetrahydrofolate; TMAO, trimethylamine N-oxide. 3979The Tdm is constituted of two domains, an uncharacterized DUF1989-containing domain at its N terminus and a tetrahydrofolate (THF)-binding domain (GCV_T) at its C-terminus. DUF1989 in Tdm shows modest sequence similarity (< 30%) to urea-carboxylase-associated proteins, whose functions in urea catabolism a...

Microbial trimethylamine metabolism in marine environments

“…Although the enzyme can be purified from aerobic bacteria, Tdm is oxygen-independent and is not affected in aerobic or anaerobic conditions (Large, 1971). This enzyme is strongly activated by Zn 2+ and Fe 2+ metal cofactors (Zhu et al, 2016).…”

Biodegradation of trimethylamine by a halotolerant strain of Paracoccus sp. PS1 and in silico analysis of trimethylamine degrading enzymes.

“…strain PS1 using the BLASTP tool. In Table S4 the aminomethyltransferase family protein of Paracoccus dentri cans was considered an outgroup as it didn't possess the conserved DUF1989 domain, which is responsible for Tdm activity (Zhu et al 2016b). The aminomethyltransferase family protein (Tdm) of strain PS1 showed conserved domains of DUF1989 and GCV_T protein which is identical to reported TMAO aldolase (Tdm) sequences of Ruegeria pomeroyi (Lidbury et al 2015) (Fig.…”

Biodegradation of trimethylamine by a halotolerant strain of Paracoccus sp. PS1 and in silico analysis of trimethylamine degrading enzymes

“…So degradation of TMAO or its precursor, TMA, using microbial enzymes in the gut may be a convenient way to reduce the detrimental effect of TMAO on human health with least side effects. The enzyme Tmm is the primary enzyme responsible for the oxidation of TMA to TMAO followed by the second enzyme, Tdm which carries out the oxygen independent demethylation of TMA into dimethylamine (DMA) and formaldehyde (Zhu et al 2016b) leading to the release of ammonia as the nal byproduct of the metabolism. This mechanism is carried out by some reported microbes such as Hyphomicrobium sp.…”