Hidden β-γ Dehydrogenation Products in Long-Chain Fatty Acid Oxidation Unveiled by NMR: Implications on Lipid Metabolism

Abstract

We present a comprehensive analysis of the initial α,β-dehydrogenation step in long-chain fatty acid β-oxidation (FAO). We focused on palmitoyl-CoA oxidized by two mitochondrial acyl-CoA dehydrogenases, very-long-chain acyl-CoA dehydrogenase (VLCAD) and acyl-CoA dehydrogenase family member 9 (ACAD9), both implicated in mitochondrial diseases. By combining MS and NMR, we identified the (2E)-hexadecenoyl-CoA as the expected α-β-dehydrogenation product and also the E and Z stereoisomers of 3-hexadecenoyl-CoA: a "γ-oxidation" product. This finding reveals an alternative catalytic pathway in mitochondrial FAO, suggesting a potential regulatory role for ACAD9 and VLCAD during fatty acid metabolism.

Scheme 1. Experimental Design and Mechanistic overview of ACAD-Mediated FAO

Superposition of VLCAD (in red) and ACAD9 (in blue) homodimers. The positions of FAD cofactor and palmitoyl-CoA (C16:0-CoA) substrate are highlighted. Inset shows key interactions between substrate and the active site. MS and NMR approaches were employed to analyze α-β and noncanonical β-γ dehydrogenation products.

Figure 1

C16:0-CoA β-oxidation was monitored by fluorescence and MS. (A) SEC traces of VLCAD and ACAD9 (black and blue lines, respectively) and SDS-PAGE showing protein monodispersity and purity. (B) AlphaFold model of the ACAD9 homodimer (in blue) in complex with ETF. (C) Mean enzyme activities for ACAD9 and VLCAD obtained from replicate assays. (D) The upper MS spectrum identifies palmitoyl-CoA in the absence of ACAD9. The lower spectrum in the presence of ACAD9 (blue) shows an additional product at 1003 Da identified as hexadecenoyl-CoA.

Figure 2

Identification of α,β and β,γ-desaturation products of C16:0-CoA by ACAD9. (A) HSQC spectrum of ¹³C₁₆-labeled C16:0-CoA (1) before incubation with ACAD9, showing resonance assignments for distinct molecular regions. Key assignments: blue for palmitic acid, purple for cysteamine, underlined for pantothenic acid, and in bold for 3′-phosphoadenosine-5′-diphosphate. Black dotted box indicates the absence of ¹H signals between 6 and 7 ppm and ¹³C signals between 120 and 155 ppm. (B) HSQC spectrum of the substrate after incubation with ACAD9 highlighting newly formed desaturation products. The red dotted box indicates the characteristic α,β-dehydrogenation product (2E)-hexadecenoyl-CoA (2, in red). In the green box: additional signals between 5.4 and 5.7 ppm in the ¹H dimension and 120–140 ppm in the ¹³C dimension suggest an alternative double bond, leading to (3E)-hexadecenoyl-CoA (3) and (3Z)-hexadecenoyl-CoA (4).

Figure 3

Structural characterization of α,β and β,γ dehydrogenation products by ACAD9. (A) Expanded region of the ¹H–¹H TOCSY spectrum, showing key correlations between protons along the acyl chain of product (2); (B) Detailed HSQC spectrum section showing the splitting of the α-proton signal into a doublet confirming the E-configuration of product (2); (C) TOCSY correlations between the protons of the new double bond in β-γ positions and the first four methylene groups of the C16:0-CoA chain after incubation with ACAD9, confirming the formation of products (3) and (4); (D) HSQC spectrum displaying two distinct cross-peaks for the allylic methylene group of products (3) and (4) indicating the presence of both E and Z isomers.

Figure 4

Alternative γ-oxidation pathway. In canonical LC-FAO (black line) ACAD catalyze the α,β-dehydrogenation of acyl-CoA to produce (2E)-enoyl-CoA, which proceeds through FAO cycle through other enzymatic steps (ECHS1 for enoyl-CoA hydratase, HADHA and HADHB for hydroxyacyl-CoA dehydrogenase alpha and beta subunits, respectively). In γ-oxidation (red line), ACAD catalyze the formation of (3E)-enoyl-CoA and (3Z)-enoyl-CoA products. These noncanonical intermediates require isomerization by enoyl-CoA isomerases (e.g., ECH1) to convert them into (2E)-enoyl-CoA, enabling entry into the canonical FAO cycle.