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[Preprint]. 2024 Feb 29:rs.3.rs-3980524.
doi: 10.21203/rs.3.rs-3980524/v1.

Structural Basis for Expanded Substrate Speci fi cities of Human Long Chain Acyl-CoA Dehydrogenase and Related Acyl- CoA Dehydrogenases

Beena Narayanan  1 , Chuanwu Xia  2 , Ryan McAndrew  1 , Anna L Shen  3 , Jung-Ja P Kim  1
Affiliations

Structural Basis for Expanded Substrate Speci fi cities of Human Long Chain Acyl-CoA Dehydrogenase and Related Acyl- CoA Dehydrogenases

Beena Narayanan et al. Res Sq. .

Update in

Abstract

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the E291Q mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.

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Conflict of interest statement

Competing interests The authors declare no competing interests.

Figures

Figures 1
Figures 1. Eucaryotic ACAD family.
The eucaryotic ACAD family includes eight ACADs for the metabolism of fatty acids with different but overlapping chain lengths (blue) and four ACADs for amino acid catabolism (yellow). ACAD9 is also involved in the mitochondrial complex 1 assembly. Electron transfer from these dehydrogenases to the main mitochondrial respiratory chain is catalyzed in sequence by electron transfer flavoprotein (ETF) and the membrane-bound ETF-ubiquinone oxidoreductase (ETF-QO), an iron-sulfur flavoprotein. Dimethylglycine dehydrogenase (DD) and sarcosine dehydrogenase (SD) are involved in choline catabolism and interact with ETF, but do not belong to the ACAD family.
Figure 2
Figure 2. The overall structure of LCAD.
a. The LCAD tetramer, or dimer of dimers (MolA:MolB and MolC:MolD dimers), is shown with monomers colored in green (MolA), cyan (MolB), magenta (MolC) and blue (MolD). FAD is shown as yellow balls and C12-CoA as gray balls. b. Representation of LCAD monomer structure with C12-CoA substrate (blue) and cofactor FAD (yellow). The red 2Fo-Fc map at 0.9σ level is derived from the E291Q LCAD crystal structure complexed with lauric acid and the gray 2Fo-Fc map at 1.0σ level is derived from the wildtype LCAD crystal structure complexed with acetoacetyl-CoA. Helices and β- sheets are labelled. Helix A(residues 54–71), B(75–83), C(86–97), D(113–128), E(133–148), F(152–164), β1 (166–172), β2(186–192), β3(194–199), β4(211–219), β5(229–237), β6(241–245), β7(258–265), Helix G(279–317), H(327–358), I(361–388), J(396–411), K(414–426).
Figure 3
Figure 3. LCAD active site.
a. Structure of E291Q LCAD complexed with C12-CoA (PDB code, 8W0Z). Hydrogen bonds are shown as dotted lines with distances indicated. FAD, Gly412, and the catalytic residue (E291Q) are shown as sticks. The C12 pro-R hydrogen and C�� pro-R hydrogen are shown in green. The 2Fo-Fc map of C12-CoA at 1.0σ level is shown in gray mesh. The distance between Cα of C12-CoA and the carboxylate of E291 is 2.8 Å and between Cβ and N5 of FAD is 3.8 Å. b. Structure of wildtype LCAD complexed with acetoacetyl-CoA, showing hydrogen bonding interactions that stabilize CoA binding. Hydrogen bonding interactions between LCAD and acetoacetyl-CoA are shown as dotted lines. The 2Fo-Fc map of acetoacetyl-CoAat 1.0σ level is shown in gray mesh. A stereo view is shown in Supplementary Figure S3.
Figure 4
Figure 4. LCAD substrate binding.
a. Surface model of LCAD substrate binding site. C12-CoA is shown in sticks with the C12 as thick sticks. FAD is shown as yellow sticks. Amino acid residues lining the substrate binding cavity are: M306, A401, L253, N206, S130, A302, V298, I295, F305, D404, Q376, V407, Y411, W121, I136, I140, I170, L294, Q408, A125; and the P-loop residues (N128, C129, S130, G131,P132, G133), are indicated. b. Surface model of LCAD substrate binding site with THC-CoA. Hydrophobic interactions between THC-CoA and residues A401, V407, A302, A125, Y411, I295, I170, I140, I136, W121, V298, L294 and Q124 are shown. Dotted lines indicate hydrogen bonding interactions between THC-CoA and the side chains of Asn128 and Tyr411 and carbonyl groups of Gln408 and Ala95.
Figure 5
Figure 5. Unwinding of the E helix is the basis for the expanded substrate cavity.
a. Overlays of the LCAD substrate binding cavity with those of SCAD (blue), MCAD (magenta), and VLCAD (cyan) are shown along with overlays of helix E (LCAD residues Phe134 to His148). The LCAD E helix is shown in green. b. Sequence alignment in the vicinity of LCAD Pro132. Note that due to Pro132, the LCAD E-helix is shorter than those of SCAD, MCAD, and VLCAD.
Figure 6
Figure 6. Stabilization of LCAD tetramer conformation by K333.
D382/K333/E336 of one LCAD monomer (MolA, green carbon atoms) and K419/E416 of another monomer (MolC, cyan atoms) form a salt bridge chain that stabilizes the tetramer conformation. Dotted lines indicate these salt bridges and a H-bond between E416 and the 3’-OH of the FAD adenosyl-ribose ring stabilizing the FAD binding.
Figure 7
Figure 7. Similarity/Conservation of LCAD, ACAD11 and MtbChsE4 substrate binding cavities.
a. LCAD substrate binding cavity (green mesh) overlaid onto ACAD11(pdb code, 2wbi) substrate binding cavity (surface model) with the substrate THC-CoA. The LCAD E-helix with Pro132 is shown in green and the corresponding ACAD11 helix with Pro463 is shown in gray. b. LCAD substrate binding cavity (green mesh) overlaid onto MtbChsE4 (pdb code, 4X08) substrate binding cavity (surface model) with the substrate THC-CoA. The LCAD E-helix with Pro132 is shown in green and the corresponding MtbChsE4 helix with Pro91 is shown in gray. c. Sequence alignment in the vicinity of LCAD Pro132.
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