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Modulating carnitine levels by targeting its biosynthesis pathway - selective inhibition of ?-butyrobetaine hydroxylase.

ABSTRACT: Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart diseases. We report the identification of potent, selective and cell active inhibitors of ?-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual 'U-shape' conformation stabilised by inter- and intra-molecular ?-stacking interactions. Conformational changes observed on binding of the inhibitor to BBOX likely reflect those occurring in catalysis; they also rationalise the inhibition of BBOX by high levels of its substrate ?-butyrobetaine (GBB), as observed both with isolated BBOX protein and in cellular studies.

SUBMITTER: Rydzik AM

PROVIDER: S-EPMC4678601 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Modulating carnitine levels by targeting its biosynthesis pathway - selective inhibition of γ-butyrobetaine hydroxylase.

Rydzik Anna M AM Chowdhury Rasheduzzaman R Kochan Grazyna T GT Williams Sophie T ST McDonough Michael A MA Kawamura Akane A Schofield Christopher J CJ

Chemical science 20140501 5

Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart diseases. We report the identification of potent, selective and cell active inhibitors of γ-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual 'U-shape' conformation stabilised by inter- and intra-m ...[more]

PMID: 26682037

Similar Datasets

Inhibition of L-carnitine biosynthesis and transport by methyl-?-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction.

Project description:Background and purposeThe important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia-reperfusion injury.Key resultsIn this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits ?-butyrobetaine dioxygenase (IC?? 3??M) and organic cation transporter 2 (OCTN2, IC?? 3??M), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5?or 20?mg·kg(-1)) decreased the infarct size by 45-48%. In vivo pretreatment with Methyl-GBB (20?mg·kg(-1)) attenuated the infarct size by 45% and improved 24?h survival of rats by 20-30%.Conclusions and implicationsReduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia-reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism.

| S-EPMC4337704 | biostudies-literature

19F NMR studies on ?-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode.

Project description:The final step in the biosynthesis of l-carnitine in humans is catalysed by the 2-oxoglutarate and ferrous iron dependent oxygenase, ?-butyrobetaine hydroxylase (BBOX). 1H and 19F NMR studies inform on the BBOX mechanism including by providing evidence for cooperativity between monomers in substrate/some inhibitor binding. The value of the 19F NMR methods is demonstrated by their use in the design of new BBOX inhibitors.

| S-EPMC6927413 | biostudies-literature

Cation-? Interactions Contribute to Substrate Recognition in ?-Butyrobetaine Hydroxylase Catalysis.

Project description:?-Butyrobetaine hydroxylase (BBOX) is a non-heme Fe(II) - and 2-oxoglutarate-dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C-H bond of ?-butyrobetaine (?BB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the ?BB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N(+) >P(+) >As(+). The results reveal that an uncharged carbon analogue of ?BB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation-? interactions in productive substrate recognition.

| S-EPMC4736438 | biostudies-literature

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

Project description: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 up-regulated 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 putative 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.

| S-EPMC8364193 | biostudies-literature

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

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| S-EPMC1144272 | biostudies-other

A conformational switch in a partially unwound helix selectively determines the pathway for substrate release from the carnitine/?-butyrobetaine antiporter CaiT.

Project description:CaiT is a homotrimeric antiporter that exchanges l-carnitine (CRN) with ?-butyrobetaine (GBB) across the bacterial membrane. Three structures have been resolved to date for CaiT, all in the inward-facing state: CRN-bound (with four CRNs per subunit), GBB-bound (two GBBs per subunit), and apo. One of the reported binding sites is the counterpart of the primary site observed in structurally similar transporters. However, the mechanism and pathway(s) of CRN/GBB unbinding and translocation, or even the ability of the substrates to dislodge from the reported binding sites, are yet to be determined. To shed light on these issues, we performed a total of 1.3 ?s of molecular dynamics simulations and examined the dynamics of substrate-bound CaiT structures under different conditions. We find that both CRN and GBB are able to dissociate completely from their primary site into the cytoplasm. Substrate molecules initially located at the secondary sites dissociate even faster (within tens of nanoseconds) into the extra- or intracellular regions. Interestingly, the unbinding pathway from the primary site appears to be dictated by the geometry of the unwound part of the transmembrane (TM) helix 3, mostly around Thr(100) therein. Arg(262) on TM7, which apparently mimics the role of Na(+) in CaiT structural homologues, plays a key role in triggering the dissociation of the substrate away from the primary site and guiding its release to the cytoplasm provided that the unwound part of TM3 switches from a shielding to a yielding pose.

| S-EPMC3442516 | biostudies-literature

?-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO.

Project description: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, ?-butyrobetaine (?BB) 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 ?BB 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 ?BB or TMA/TMAO from dietary L-carnitine. Moreover, despite their close structural similarity, chronic dietary exposure to L-carnitine or ?BB promotes development of functionally distinct microbial communities optimized for the metabolism of L-carnitine or ?BB, respectively.

| S-EPMC4255476 | biostudies-literature

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

Project description: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.

2021-02-03 | GSE165976 | GEO

Selective Inhibition of Collagen Prolyl 4-Hydroxylase in Human Cells.

Project description:Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4-hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a "P4H" inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2'-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2-(5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents.

| S-EPMC4798942 | biostudies-literature

treatment g-butyrobetaine wt col.0-Effect of carnitine on the transcriptome of A. thaliana

Project description:rs07-01_carnitine - treatment g-butyrobetaine wt col.0 - Effect of carnitine on the transcriptome of A. thaliana - Seedlings WT are growing in a independent way on media MS. After seven days of culture, seedling are harvested and a pull is formed from the three prelevements. Extractions of RNA will be realised on this pull. We proceed in a identical way for the test sample. Seedlings are cultivated on media MS containing butyrobétaïne 1 mM. After seven days, seedlings are harvested and extractions of RNA will be realised on this second pull. All this experiment is repeated one more time independently. Keywords: treated vs untreated comparison

2009-02-18 | GSE10677 | GEO

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