Publications

• Biochemical and structural elucidation of the L-carnitine degradation pathway of the human pathogen <i>Acinetobacter baumannii</i>.

  Piskol F., Lukat P., Kaufhold L., Heger A., Blankenfeldt W., Jahn D., Moser J.

  <i>Acinetobacter baumannii</i> is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. S ... >> More

  <i>Acinetobacter baumannii</i> is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. Subsequent conversion of the resulting malic semialdehyde into the central metabolite L-malate was hypothesized. Alternatively, L-carnitine degradation via D-malate with subsequent oxidation into pyruvate was proposed. Here we describe the <i>in vitro</i> and <i>in vivo</i> reconstitution of the entire pathway, starting from the as yet uncharacterized gene products of the carnitine degradation gene operon. Using recombinantly purified enzymes, enantiomer-specific formation of D-malate by the NAD(P)⁺-dependent malic semialdehyde dehydrogenase (MSA-DH) is demonstrated. The solved X-ray crystal structure of tetrameric MSA-DH reveals the key catalytic residues Cys²⁹⁰ and Glu²⁵⁶, accessible through opposing substrate and cofactor funnels. Specific substrate binding is enabled by Arg¹⁶⁶, Arg²⁸⁴ and Ser⁴⁴⁷ while dual cofactor specificity for NAD⁺ and NADP⁺ is mediated by Asn¹⁸⁴. The subsequent conversion of the unusual D-malate reaction product by an uncharacterized NAD⁺-dependent malate dehydrogenase (MDH) is shown. Tetrameric MDH is a β-decarboxylating dehydrogenase that synthesizes pyruvate. MDH experiments with alternative substrates showed a high degree of substrate specificity. Finally, the entire <i>A. baumannni</i> pathway was heterologously reconstituted, allowing <i>E. coli</i> to grow on L-carnitine as a carbon and energy source. Overall, the metabolic conversion of L-carnitine via malic semialdehyde and D-malate into pyruvate, CO₂ and trimethylamine was demonstrated. Trimethylamine is also an important gut microbiota-dependent metabolite that is associated with an increased risk of cardiovascular disease. The pathway reconstitution experiments allowed us to assess the TMA forming capacity of gut microbes which is related to human cardiovascular health. << Less

  Front Microbiol 15:1446595-1446595(2024) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Gamma-butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO.

  Koeth R.A., Levison B.S., Culley M.K., Buffa J.A., Wang Z., Gregory J.C., Org E., Wu Y., Li L., Smith J.D., Tang W.H., DiDonato J.A., Lusis A.J., Hazen S.L.

  L-carnitine, a nutrient in red meat, was recently reported to accelerate atherosclerosis via a metaorganismal pathway involving gut microbial trimethylamine (TMA) formation and host hepatic conversion into trimethylamine-N-oxide (TMAO). Herein, we show that following L-carnitine ingestion, γ-butyr ... >> More

  L-carnitine, a nutrient in red meat, was recently reported to accelerate atherosclerosis via a metaorganismal pathway involving gut microbial trimethylamine (TMA) formation and host hepatic conversion into trimethylamine-N-oxide (TMAO). Herein, we show that following L-carnitine ingestion, γ-butyrobetaine (γBB) is produced as an intermediary metabolite by gut microbes at a site anatomically proximal to and at a rate ∼1,000-fold higher than the formation of TMA. Moreover, we show that γBB is the major gut microbial metabolite formed from dietary L-carnitine in mice, is converted into TMA and TMAO in a gut microbiota-dependent manner (like dietary L-carnitine), and accelerates atherosclerosis. Gut microbial composition and functional metabolic studies reveal that distinct taxa are associated with the production of γBB or TMA/TMAO from dietary L-carnitine. Moreover, despite their close structural similarity, chronic dietary exposure to L-carnitine or γBB promotes development of functionally distinct microbial communities optimized for the metabolism of L-carnitine or γBB, respectively. << Less

  Cell Metab. 20:799-812(2014) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• CntA oxygenase substrate profile comparison and oxygen dependency of TMA production in Providencia rettgeri.

  Kalnins G., Sevostjanovs E., Hartmane D., Grinberga S., Tars K.

  CntA oxygenase is a Rieske 2S-2Fe cluster-containing protein that has been previously described as able to produce trimethylamine (TMA) from carnitine, gamma-butyrobetaine, glycine betaine, and in one case, choline. TMA found in humans is exclusively of bacterial origin, and its metabolite, trimet ... >> More

  CntA oxygenase is a Rieske 2S-2Fe cluster-containing protein that has been previously described as able to produce trimethylamine (TMA) from carnitine, gamma-butyrobetaine, glycine betaine, and in one case, choline. TMA found in humans is exclusively of bacterial origin, and its metabolite, trimethylamine oxide (TMAO), has been associated with atherosclerosis and heart and renal failure. We isolated four different Rieske oxygenases and determined that there are no significant differences in their substrate panels. All three had high activity toward carnitine/gamma-butyrobetaine, medium activity toward glycine betaine, and very low activity toward choline. We tested the influence of low oxygen concentrations on TMA production in CntA-containing Providencia rettgeri cell cultures and discovered that this process, although dependent on the amount of oxygen, is still feasible in environments with 1 and 0.2% oxygen, which is comparable to oxygen levels in some parts of the digestive system. << Less

  J Basic Microbiol 58:52-59(2018) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer.

  Quareshy M., Shanmugam M., Townsend E., Jameson E., Bugg T.D.H., Cameron A.D., Chen Y.

  Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase famil ... >> More

  Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α₃ trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 Å apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut. << Less

  J Biol Chem 296:100038-100038(2021) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.