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. 2013 Nov 22;288(47):33837-33847.
doi: 10.1074/jbc.M113.510354. Epub 2013 Oct 11.

Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site

Sivakama S Bharathi  1 , Yuxun Zhang  1 , Al-Walid Mohsen  1 , Radha Uppala  1 , Manimalha Balasubramani  2 , Emanuel Schreiber  2 , Guy Uechi  2 , Megan E Beck  3 , Matthew J Rardin  4 , Jerry Vockley  5 , Eric Verdin  6 , Bradford W Gibson  4 , Matthew D Hirschey  7 , Eric S Goetzman  8
Affiliations

Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site

Sivakama S Bharathi et al. J Biol Chem. .

Abstract

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.

Keywords: Acyl-CoA Dehydrogenase; Electron Transfer; Enzyme Catalysis; FAD; Fatty Acid Oxidation; Lysine Acetylation; Mitochondria; Posttranslational Modification; Sirtuins.

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Figures

FIGURE 1.
FIGURE 1.
Chemically acetylated recombinant LCAD is a substrate for SIRT3. a, anti-acetyllysine Western blotting of acetic anhydride (Anh)- versus mock-treated recombinant LCAD protein, 20 ng/lane. Ctrl, control. b, 2.0 μm chemically acetylated LCAD was deacetylated by SIRT3 for the indicated time periods. Deacetylation was measured by following degradation of [14C]NAD+. Data points represent the average of duplicate assays. c, chemically acetylated LCAD and BSA were used as substrates for in vitro deacetylation assays with recombinant human SIRT3. Data points represent the average of duplicate assays. conc., concentration. d, anhydride acetylation of LCAD significantly reduces enzymatic activity. Deacetylation of anhydride-treated LCAD with SIRT3, but not the inactive mutant SIRT3-H248Y, partially restores enzymatic activity. Error bars represent mean ± S.D. of triplicate assays. e and f, sulfo-NHS-acetate was tested as an alternative to anhydride, which is chemically unstable. Incubation of LCAD with increasing concentrations of sulfo-NHS-acetate both increased the rate of SIRT3 deacetylation (e) and reduced the enzymatic activity of LCAD (f). Subsequent experiments utilized sulfo-NHS-acetate as an acetylating agent.
FIGURE 2.
FIGURE 2.
Acetylation affects the active site of LCAD. Recombinant LCAD was either mock-treated (a) or acetylated with sulfo-NHS-acetate (Ac-LCAD) (b) and then titrated with palmitoyl-CoA under anaerobic conditions to study the reductive half-reaction. After each addition of substrate, the enzyme was allowed to stabilize for 10 s and then scanned on a spectrophotometer from 300–800 nm. The characteristic FAD peak (444 nm) becomes reduced, and the charge-transfer complex peak (∼570 nm) increases with increasing substrate concentrations. c, the absorbance (Abs.) of the FAD peak (444 nm) from the curves in a and b were plotted, fit with nonlinear regression, and used to calculate Δmax (maximum change in absorbance) and Kf (apparent substrate binding constant, in micromolar). d, the oxidative half-reaction was studied by kinetic assays with increasing concentrations of the physiological electron acceptor ETF. Data points represent the average of duplicate assays. The curves were fit with non-linear regression (Michaelis-Menten) and used to calculate Km, Vmax, and catalytic efficiency.
FIGURE 3.
FIGURE 3.
LCAD residues Lys-318 and Lys-322 are targets of SIRT3 and modulate enzymatic activity. a, chemically acetylated LCAD was either mock-treated or SIRT3-treated in quadruplicate. The protein samples were digested with trypsin, labeled with 8-plex iTRAQ isobaric tags, and subjected to LC-MS/MS. The relative abundance of eight peptides changed significantly with SIRT3 incubation. Three of these were acetylated peptides (Lys-42, Lys-318, and Lys-322), and five were unmodified peptides. Some peptides with non-significant change are shown for context. *, p < 0.05; SIRT3-treated versus control; N/A, not applicable, this peptide was not quantified in the iTRAQ assay. b, the three lysines identified by iTRAQ proteomics (Lys-42, Lys-318, and Lys-322) were mutated to either alanine (A) or arginine (R) and expressed in E. coli. Crude lysates (5 μg) were Western-blotted with anti-LCAD antibody. 10 ng of purified LCAD was run as positive control (PC). Subsequently, six of these mutant proteins were purified to homogeneity (K42R, K318A, K318R, K322A, K322R, and K318R/K322R), and enzymatic activity was measured in triplicate. Activities are shown as a percentage of WT LCAD activity below the Western blot. N/A, not applicable. These proteins were not purified and thus were not assayed. c, the purified arginine mutant LCAD proteins were acetylated with sulfo-NHS-acetate and used as substrates for SIRT3 deacetylation assays. Shown is the mean ± S.D. of triplicate 20-min deacetylation assays using 19 μm LCAD monomer. *, p < 0.001 versus the wild type. d, samples of wild-type LCAD and arginine mutants were acetylated with sulfo-NHS-acetate and tested for enzymatic activity versus mock-treated enzyme. For ease of comparison, the activity of each mock-treated enzyme was set to 1.0. Note that only K318R/K322R had reduced starting activity compared with WT (b). Shown is the mean ± S.D. of triplicate activity assays. *, p < 0.001, acetylated enzyme versus the corresponding non-acetylated control; #, p < 0.001 acetylated mutant LCAD versus acetylated wild-type LCAD.
FIGURE 4.
FIGURE 4.
Acetylation at Lys-318 and Lys-322 is predicted to alter the conformation of the LCAD active site. a, amino acid sequence alignment of the region containing Lys-318 and Lys-322. All ACADs contain an invariant arginine (position 317 in LCAD) and phenylalanine (position 320 in LCAD). Mouse LCAD, human LCAD, human MCAD, and human ACAD9 have lysines at the positions equivalent to 318 and 322. Mouse MCAD and ACAD9 also have lysines at these positions (not shown). Other species besides mouse and human were not examined. VLCAD, very long-chain acyl-CoA dehydrogenase; SCAD, short-chain acyl-CoA dehydrogenase. b, ribbon and stick representation of the interface of LCAD and ETF. The model was generated from the published atomic coordinates of MCAD complexed with ETF (PDB code 2A1T, Ref. 30). The ribbon shown in jade is for LCAD monomer A of the tetramer, whereas the white ribbon is for LCAD monomer B. The tan ribbon is for the α subunit of ETF. The substrate modeled and shown is an octanoyl-CoA. c, purified mutant enzymes with substitutions at Lys-318 and Lys-322 were evaluated for FAD content using a commercial kit. Shown is the mean ± S.D. of triplicate assays. *,p < 0.001 versus wild-type LCAD.
FIGURE 5.
FIGURE 5.
LCAD Lys-318 is a SIRT3 target site in vivo, whereas Lys-81 and Lys358 do not appear to play a role in the regulation of LCAD activity by acetylation. a, SRM proteomics were used to determine the abundance of the acetylated peptide KAFGK (acetylated at Lys-318) in liver mitochondria isolated from wild-type and SIRT3 knockout mice (n = 5). As a control, the abundance of the acetylated peptide LETPSAKK was determined. Samples were supplemented with isotopically labeled LETPSAKK* (acetylated at Lys-42, and where the C-terminal residue is 13C6,15N2-lysine), and the resulting heavy peptide signal was used for normalization. Shown is the mean ± S.D. *, p < 0.05. b and c, the importance of Lys-81 and Lys-358 as potential SIRT3-targeted sites was evaluated by creating K81R and K358R mutant LCAD proteins, chemically acetylating them with sulfo-NHS-acetate, and testing the resulting acetylated proteins as substrates for SIRT3 (b) and for enzymatic activity (c). The K81R and K358R substitutions did not affect the rate of deacetylation or provide protection against acetylation-induced loss of activity. Shown is the mean ± S.D. of triplicate assays. *, p < 0.001 for acetylated versus control proteins.
FIGURE 6.
FIGURE 6.
Chemically acetylated MCAD and ACAD9 are also substrates for SIRT3. a–c, purified MCAD, ACAD9, and IVD were chemically acetylated with sulfo-NHS-acetate and used as substrates in kinetic deacetylation assays with purified SIRT3. MCAD and ACAD9, which have lysines at positions equivalent to Lys-318/Lys-322, both displayed Michaelis-Menten kinetics with SIRT3, whereas IVD, which does not have lysines at these positions, was not saturable out to 55 μm protein concentration. Each data point represents the average of duplicate assays.
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