Elucidation of an anaerobic pathway for metabolism of L-carnitine-derived γ-butyrobetaine to trimethylamine in human gut bacteria

Abstract

Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient L-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent L-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the L-carnitine-derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis. We identify a set of genes upregulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a flavin cofactor to catalyze a redox neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from L-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of L-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.SIGNIFICANCETrimethylamine (TMA) is a disease-associated metabolite produced in the human body exclusively by microbes. Gut microbes generate TMA from essential nutrients consumed in the human diet, including L-carnitine. However, our understanding of the biochemical mechanisms involved in these transformations is incomplete. In this work, we define the biochemical pathway and genetic components in gut bacteria required for anaerobic production of TMA from γ-butyrobetaine, a metabolite derived from L-carnitine. This discovery identifies a new type of TMA-producing enzyme and fills a critical gap in our knowledge of L-carnitine metabolism to TMA in the anaerobic environment of the human gut. This knowledge will enable evaluation of the link between L-carnitine metabolism and human disease, and the design of potential therapeutics.

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase

“…Both the oxygenases, YeaWX ( 15 ) and CntAB ( 32 , 37 ), have been shown to cleave γ-butyrobetaine to TMA. However, it is not clear if these enzymes function to a significant extent in the largely anoxic intestine ( 38 ). Only recently was BbuA described, a flavoprotein that functions anoxically to cleave γ-butyrobetainyl-CoA to crotonyl-CoA and TMA ( 38 ).…”

“…However, it is not clear if these enzymes function to a significant extent in the largely anoxic intestine ( 38 ). Only recently was BbuA described, a flavoprotein that functions anoxically to cleave γ-butyrobetainyl-CoA to crotonyl-CoA and TMA ( 38 ). Highly similar homologs of bbuA are found in intestinal isolates as well as gut microbiota metagenomic datasets ( 38 ).…”

“…Only recently was BbuA described, a flavoprotein that functions anoxically to cleave γ-butyrobetainyl-CoA to crotonyl-CoA and TMA ( 38 ). Highly similar homologs of bbuA are found in intestinal isolates as well as gut microbiota metagenomic datasets ( 38 ). The rise in TMAO levels after eating red meat is attributable to the l -carnitine/γ-butyrobetaine pathway ( 39 ).…”

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“…Similarly, γ-butyrobetaine undergoes metabolism by either the MtyB/MtqAC methyltransferase (Rxn 2a) described by Ellenbogen et al. ( 3 ) or BbuABC (Rxn 2b) ( 7 ) for generation of CH 3 -H 4 folate or TMA. Entry of TMA into the blood stream, where a host monooxygenase catalyzes its oxidation to TMAO (Rxn 3b), is counterbalanced by microbial MttB-catalyzed conversion to CH 3 -CoM (Rxn 3a) ( 10 ).…”

“…As shown in Figure 1 , consumption of γ-butyrobetaine by MtyB and of carnitine by MtcB by E. limosum and other microorganisms in the microbiome might provide a natural competitive mechanism to lower the production of proatherogenic metabolites TMA and TMAO. TMA is produced predominantly by the recently discovered anaerobic bbu pathway (BbuABCDEF) ( 7 ), with TMAO generated by a host monooxygenase. On the other hand, the methyl transfer reactions produce methyl-H 4 folate, which couples directly to the Wood–Ljungdahl pathway for the synthesis of acetyl-CoA ( 8 ), which in turn is linked to biomass generation and energy conservation for the bacteria as well as the synthesis of beneficial short-chain fatty acids.…”