TY - JOUR

T1 - Identification and characterization of trimethylamine N-oxide (TMAO) demethylase and TMAO permease in Methylocella silvestris BL2

AU - Zhu, Yijun

AU - Jameson, Eleanor

AU - Parslow, Rosemary A

AU - Lidbury, Ian

AU - Fu, Tiantian

AU - Dafforn, Timothy R

AU - Schäfer, Hendrik

AU - Chen, Yin

N1 - © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

PY - 2014/10/1

Y1 - 2014/10/1

N2 - Methylocella 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 Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting of an N-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO → 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Tdm of M. silvestris and eukaryotic Tdms have no sequence homology and contrasting characteristics. Recombinant Tdm of M. silvestris appears to be hexameric, has a high affinity for TMAO (Km = 3.3 mM; Vmax = 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.

AB - Methylocella 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 Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting of an N-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO → 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Tdm of M. silvestris and eukaryotic Tdms have no sequence homology and contrasting characteristics. Recombinant Tdm of M. silvestris appears to be hexameric, has a high affinity for TMAO (Km = 3.3 mM; Vmax = 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.

KW - Aldehyde-Lyases/genetics

KW - Alphaproteobacteria/enzymology

KW - Escherichia coli/genetics

KW - Genes, Bacterial

KW - Membrane Transport Proteins/genetics

KW - Methylamines/metabolism

KW - Mutagenesis

U2 - 10.1111/1462-2920.12585

DO - 10.1111/1462-2920.12585

M3 - Article

C2 - 25088783

VL - 16

SP - 3318

EP - 3330

JO - Environmental Microbiology

JF - Environmental Microbiology

SN - 1462-2920

IS - 10

ER -