Identification and characterization of trimethylamine N ‐oxide ( TMAO ) demethylase and TMAO permease in M ethylocella silvestris   BL 2

• Published in: Environmental microbiology 2014-10, Vol.16 (10), p.3318-3330
• Main Authors: Zhu, Yijun, Jameson, Eleanor, Parslow, Rosemary A., Lidbury, Ian, Fu, Tiantian, Dafforn, Timothy R., Schäfer, Hendrik, Chen, Yin
• Format: Article
• Language: English
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/1462-2920.12585

M ethylocella silvestris , an alphaproteobacterium isolated from a forest soil, can grow on trimethylamine N ‐oxide ( TMAO ) as a sole nitrogen source; however, the molecular and biochemical mechanisms underpinning its growth remain unknown. Marker‐exchange mutagenesis enabled the identification of several genes involved in TMAO metabolism, including M sil_3606 , a permease of the amino acids‐polyamine ( APC ) superfamily, and M sil_3603 , consisting of an N ‐terminal domain of unknown function ( DUF 1989) and a C ‐terminal tetrahydrofolate‐binding domain. Null mutants of M sil_3603 and M sil_3606 can no longer grow on TMAO . Purified M sil_3603 from recombinant E scherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO  → 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase ( T dm). T dm of M . silvestris and eukaryotic T dms have no sequence homology and contrasting characteristics. Recombinant T dm of M . silvestris appears to be hexameric, has a high affinity for TMAO ( K m = 3.3  mM ; V max = 21.7 nmol min −1  mg −1 ) and only catalyses demethylation of TMAO and a structural homologue, dimethyldodecylamine N ‐oxide. Our study has contributed to the understanding of the genetic and biochemical mechanisms for TMAO degradation in M . silvestris .