Mammalian hydroxylation of microbiome-derived obesogen, delta-valerobetaine, to homocarnitine, a 5-carbon carnitine analog

Abstract

The recently discovered microbiome-generated obesogen, δ-valerobetaine (5-(trimethylammonio)pentanoate), is a 5-carbon structural analog of the carnitine precursor, γ-butyrobetaine. Here, we report that δ-valerobetaine is enzymatically hydroxylated by mammalian γ-butyrobetaine dioxygenase (BBOX) to form 3-hydroxy-5-(trimethylammonio)pentanoate, a 5-carbon analog of carnitine, which we term homocarnitine. Homocarnitine production by human liver extracts depends upon the required BBOX cofactors, 2-oxoglutarate, Fe²⁺, and ascorbate. Molecular dynamics simulations show successful docking of δ-valerobetaine and homocarnitine to BBOX, pharmacological inhibition of BBOX prevents homocarnitine production, and transfection of a liver cell line with BBOX substantially increases production. Furthermore, an in vivo isotope tracer study shows the conversion of ¹³C₃-trimethyllysine to ¹³C₃-δ-valerobetaine then ¹³C₃-homocarnitine in mice, confirming the in vivo production of homocarnitine. Functional assays show that carnitine palmitoyltransferase acylates homocarnitine to acyl-homocarnitine, analogous to the reactions for the carnitine shuttle. Studies of mouse tissues and human plasma show widespread distribution of homocarnitine and fatty acyl-homocarnitines. The respective structural similarities of homocarnitine and acyl-homocarnitines to carnitine and acyl-carnitines indicate that homocarnitine could impact multiple sites of carnitine distribution and activity, potentially mediating microbiome-associated obesity and metabolic disorders.

Keywords: BBOX; acyltransferase; carnitine; energy metabolism; fatty acid metabolism; homocarnitine; microbiome; obesity; δ-valerobetaine.

Figure 1

Scheme showing structures of γ-butyrobetaine and its BBOX-generated product, carnitine, and δ-valerobetaine with its analogous BBOX-generated product, homocarnitine.

Figure 2

Molecular dynamics simulations of BBOX1 interactions with δ-valerobetaine and homocarnitine. Modeling studies conducted with Maestro and Glide to dock γ-butyrobetaine and its BBOX-generated product, carnitine, and δ-valerobetaine and its proposed BBOX-generated product, homocarnitine in the catalytic site of BBOX1 (PDB: 3O2G (17)). Residues with > 30% interaction times across three simulations are shown. Interaction times between molecule atoms and BBOX residues are represented as the average percentage of three simulations that contacts are made. All key residues with interaction fraction times and indicated chemical bond type are available in Fig. S2. Green = carbon atoms on substrate or product. Red atoms = oxygen. Blue atoms = nitrogen. Red spheres = water. Red dashed lines = hydrogen bond. Purple dashed lines = ionic interaction. Green dashed lines = cation-π interaction.

Figure 3

Bioconversion of δ-valerobetaine to homocarnitine. A, intensity of m/z 176.1281 corresponding to homocarnitine in mouse liver S9 fractions incubated with 50 μM δ-valerobetaine and either vehicle (black) or BBOX cofactors (red) (N = 3). BBOX cofactors consisted of 3 mM 2-oxoglutarate, 0.25 mM ferrous chloride, and 10 mM sodium ascorbate. Means and standard error of the mean (SEM) are shown. B, homocarnitine production (m/z 176.1281) in primary rat hepatocytes incubated for 6 h with vehicle or 100 μM δ-valerobetaine with or without 100 μM meldonium, a competative BBOX inhibitor. C, homocarnitine (m/z 176.1281) production by Huh7 and stably transfected BBOX^Ala-Huh7 and BBOX-Huh7 cell lines incubated for 4 h with 100 μM δ-valerobetaine. D, extracted ion chromatogram from HILIC/ESI+ analysis of BBOX-Huh7 cell lysate shows coelution of homocarnitine (m/z 176.1281) (black) with synthetic 10 μM ¹³C₃-homocarnitine (m/z 179.1383) (red). E, mirror plot showing MS² ion dissociation spectra of homocarnitine (HCD 35%) from BBOX-Huh7 cell lysate (black; top) and synthetic homocarnitine at 10 μM in saline extracted by acetonitrile (red; bottom). On the right, proposed structures and exact masses (Da) of observed product ions (A: 117.0546, B = 99.0441, C = 60.0809) resulting from 2 fragmentation sites on homocarnitine’s structure (red lines) are presented; note that m/z 99.0441 could also form a cyclic structure. Group differences in panels B and C were determined with a one-way ANOVA followed by Tukey’s test of multiple comparisons (p ≤ 0.001 = ∗∗∗, ns = p > 0.05).

Figure 4

Isotope tracer study of homocarnitine formation in mice.A, extracted ion chromatogram from HILIC/ESI+ for homocarnitine (m/z 176.1281) in urine of mice gavaged with 100 mg/kg trimethyllysine (TML) coelutes with ¹³C₃-homocarnitine (m/z 179.1383) in urine of mice gavaged with 25 mg/kg ¹³C₃-trimethyllysine (¹³C₃-TML). B, MS² ion dissociation (HCD 25%) spectrum of m/z 176.1281 in mouse urine shows m/z 60.0809 and m/z 99.0441 product ions from homocarnitine. C, MS² ion dissociation (HCD 25%) spectrum of m/z 179.1383 for ¹³C₃-homocarnitine in urine of mice given ¹³C₃-TML shows corresponding product ion m/z 63.0909 representing the labelled carbons and other homocarnitine product ion at m/z 99.0441. Relative tissue abundances of labelled ¹³C₃-homocarnitine (m/z 179.1383) from the tracer study are provided for (D) urine, (E) liver, and (F) serum.

Figure 5

Effectof δ-valerobetaine on tissue levels of homocarnitine and serum carnitine metabolites. The effect of oral δ-valerobetaine dose on relative abundance of homocarnitine in mouse heart (A), liver (B), serum (C), and brain (D) with means shown. E, serum levels of carnitine (m/z 162.1125) at each dose of δ-valerobetaine. F, relative intensities of spectral features are indicated from low (blue) to high (red) across 5 mouse replicates treated with saline or 100 mg/kg δ-valerobetaine. Relative intensities were calculated individually for each metabolite in each row. Female data are presented; male mice showed similar response (data not shown). Group differences in panels (A–E) were determined with a one-way ANOVA followed by Tukey’s test of multiple comparisons (p ≤ 0.05 = ∗, p ≤ 0.01 = ∗∗, p ≤ 0.001 = ∗∗∗, ns = p > 0.05).

Figure 6

Formation and detection of fatty acyl-homocarnitines.A, scheme showing CPT1-catalyzed fatty acylation of homocarnitine produced from δ-valerobetaine by BBOX and CPT1 inhibition by etomoxir. B, levels of ¹³C₁₆-palmitoyl-homocarnitine (m/z 430.4106) in BBOX-Huh7 cells preincubated with 200 μM δ-valerobetaine then treated with 40 μM [¹³C₁₆]palmitate plus vehicle or 40 μM etomoxir (p < 0.001). C, extracted ion chromatogram from HILIC/ESI+ shows coelution of palmitoyl-homocarnitine (m/z 414.3578) and ¹³C₁₆-palmitoyl-homocarnitine (m/z 430.4106) in BBOX-Huh7 cells treated with δ-valerobetaine and [¹³C₁₆]palmitate. D, MS² ion dissociation of ¹³C₁₆-palmitoyl-homocarnitine (m/z 430.4106) in BBOX-Huh7 cells treated with δ-valerobetaine and [¹³C₁₆]palmitate shows expected product ions. Proposed structures for observed ¹³C₁₆-palmitoyl-homocarnitine product ions are described in Fig. S6. E, extracted ion chromatogram of ¹³C₂-acetyl-homocarnitine (m/z 220.1454) and acetyl-homocarnitine (m/z 218.1387) in BBOX-Huh7 cells treated with δ-valerobetaine and [¹³C₁₆]palmitate. F, MS² ion dissociation of ¹³C₂-acetyl-homocarnitine (m/z 220.1454) in BBOX-Huh7 cells treated with δ-valerobetaine and [¹³C₁₆]palmitate shows expected product ions. Proposed structures for observed ¹³C₂-acetyl-homocarnitine product ions are described in Fig. S7. Chromatograms and MS² spectra for in vivo heart palmitoyl-homocarnitine (m/z 414.3578) and acetyl-homocarnitine (m/z 218.1387) are provided in Fig. S9; proposed structures for respective product ions are provided in Figs. S6 and S10. Results from a similar experiment conducted in primary rat hepatocytes with pharmacological CPT1 and BBOX inhibition is available in Fig. S8.

Figure 7

Detection of acetyl-homocarnitine in human samples.A, extracted ion chromatogram of homocarnitine in a pooled reference sample of human plasma. B, MS² ion dissociation of homocarnitine in a pooled reference sample of human plasma shows product ions characteristic of homocarnitine (see Fig. 3E). C, Spearman correlation (r_s = 0.8, p < 0.001) of δ-valerobetaine and homocarnitine in human urine (N = 165). D, extracted ion chromatogram of the isomeric species acetyl-homocarnitine and propionyl-carnitine (m/z 218.1387) in a pooled reference sample of human plasma. E, MS² ion dissociation spectrum of the first peak representing acetyl-homocarnitine (m/z 218.1387; retention time (RT) = 0.6 min) with expected product ions (m/z 99.0436, 158.1178, 172.1332) in a pooled reference sample of human plasma. F, MS² ion dissociation spectrum of the second peak representing propionyl-carnitine (m/z 218.1387; RT = 0.9 min) with expected product ions (m/z 60.0812, 85.0286, 159.0657) in a pooled reference sample of human plasma. The intensity of product ion m/z 144.1022 for propionyl-carnitine is also detectable but low in this spectrum. Relative intensity of this product ion was higher in other samples. Estimated concentrations of homocarnitine in human plasma and urine are provided in Fig. S11. Correlation of plasma δ-valerobetaine with homocarnitine is provided in Fig. S12. Proposed structures of acetyl-homocarnitine and propionyl-carnitine product ions are available in Fig. S10.

Figure 8

Proposed metabolic pathway of homocarnitine synthesis. (Left) Prior research shows that mammalian enzymes produce γ-butyrobetaine from trimethyllysine (71, 72) through a sequence of hydroxylation (73, 74), cleavage (75), and dehydrogenation (76) reactions. (Right) Recent studies identified two bacterial enzymes in the intestinal microbiome, lysine 2-monooxygenase (DavB; EC 1.13.12.2) and 5-aminovaleramide amidohydrolase (DavA; EC 3.5.1.30), that metabolize trimethyllysine to δ-valerobetaine (2, 21, 22). It was previously established that these two enzymes metabolize lysine to 5-aminovaleramide then to 5-aminovalerate. Similarly, lysine 2-monooxygenase converts trimethyllysine to trimethyl-5-aminovaleramide which is then converted to δ-valerobetaine by 5-aminovaleramide amidohydrolase (2, 22, 77, 78, 79). The present research shows that δ-valerobetaine is hydroxylated to form homocarnitine in a subsequent step catalyzed by mammalian γ−butyrobetaine dioxygenase (BBOX) (12, 80, 81), the enzyme that converts γ−butyrobetaine to carnitine. Enzymes are listed in blue (mammalian) and green (bacterial) boxes. Created with BioRender.com.

Figure 9

Proposed metabolic pathway of homocarnitine acylation. The present research shows that homocarnitine is acylated to form palmitoyl-homocarnitine, with pharmacologic evidence that this is catalyzed by carnitine palmitoyl transferase-1 (CPT1). CPT1 is an enzyme that exists in different tissue-specific forms and catalyzes fatty acyl-carnitine formation from acyl-CoA’s and carnitine (4, 82). Carnitine-acylcarnitine translocase (CACT) (83, 84) transports free and acylated-carnitines into the mitochondrial matrix where CPT2 (50) replaces the carnitine on a fatty acyl chain with a CoA to prepare for β-oxidation. Similarly, the present research shows existence of acetyl-homocarnitine likely indicating that homocarnitine and/or palmitoyl-homocarnitine can enter the mitochondria via CACT. The final product of β-oxidation is acetyl-CoA which can be converted to acetyl-carnitine or acetyl-homocarnitine by carnitine acetyltransferase (CAT or CrAT) (52). Created with BioRender.com. OM, outer mitochondrial membrane; IM,: inner mitochondrial membrane.