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. 2014 Mar 18;111(11):4268-73.
doi: 10.1073/pnas.1316569111. Epub 2014 Mar 3.

Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Yijun Zhu  1 , Eleanor Jameson, Marialuisa Crosatti, Hendrik Schäfer, Kumar Rajakumar, Timothy D H Bugg, Yin Chen
Affiliations

Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Yijun Zhu et al. Proc Natl Acad Sci U S A. .

Abstract

Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate intersubunit electron transfer between the Rieske center and the catalytic mononuclear iron center. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Taken together, the data in our study reveal the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assign the role of this novel group of Rieske-type proteins in microbial carnitine metabolism.

Keywords: comparative genomics; gut microbiota; methylated amine metabolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of the putative Rieske-type protein from human microbiota in carnitine-dependent TMA formation. (A) Putative carnitine-to-TMA gene cluster in representative genomes of human microbiota. CaiT, carnitine transporter; CntA, A Rieske-type oxygenase protein; CntB, a predicted reductase with a plant-type ferridoxin domain. (B) Analyses of conserved domains in CntA and CntB. FAD: flavin adenine dinucleotide; NAD+: nicotinamide adenine dinucleotide. (C) An unrooted phylogenetic tree (∼305 aa) of CntA, microbial Rieske-type terminal oxygenases (groups I–IV) and eukaryotic Rieske-type choline monooxygenases. Microbial Rieske-type terminal oxygenases (groups I–IV) are classified based on the nomenclature system of Nam et al. (57). These sequences are identified by a unique GenBank or PDB accession number followed by the gene name; other sequences are identified by the name of the species. Bootstrap values greater than 50 are shown (100 replicates). The scale bar represents one substitution per 10 amino acids. (D) Predicted pathway of carnitine catabolism via CntA/B.
Fig. 2.
Fig. 2.
Growth of A. baumannii ATCC19606 wild-type, mutants (∆cntA::aacC1, ∆cntB::aacC1), and complemented mutants with plasmid pKR706 on carnitine (10 mM) or succinate (20 mM) as the sole carbon and energy source. The error bars represent SD from experiments run in eight replicates.
Fig. 3.
Fig. 3.
Quantification of TMA and carnitine of wild-type, mutants (∆cntA::aacC1, ∆cntB::aacC1), and complemented mutants with plasmid pKR706 in the culture medium supplemented with carnitine and succinate. The error bars represent SD from experiments run in triplicate.
Fig. 4.
Fig. 4.
Quantification of TMA formation in vitro using the supernatant of recombinant E. coli containing overexpressed CntA, CntB, or CntAB, respectively. The error bars represent SD from experiments run in triplicate.
Fig. 5.
Fig. 5.
The unusual substitution of “bridging” aspartate to “bridging” glutamate (E205 in CntA) abolished catalytic activity for carnitine-dependent TMA formation. (A) Multiple sequence alignment of CntA from representative human microbiota and related Rieske-type proteins. The two boxes highlight the conserved Rieske domain and the mononuclear iron center domain. The arrow indicates the unusual but conserved glutamate residue in CntA. Each sequence has a unique identifiable label as shown in the legend of Fig. 1C. (B) Quantification of NADH oxidation and TMA production in vitro using purified CntA and site-directed mutants (E205A, E205D) in combination with purified CntB. Coupling efficiency is determined as the ratio of the total amount of TMA formed to the amount of NADH consumed. The error bars indicate SD from experiments run in triplicate.
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