Sirt5 catalyzed the hydrolysis of malonyl and succinyl lysine. The enzymatic reactions were analyzed by LC-MS. Pink traces showed the ion intensities (10× magnified) for the masses of the acyl peptides (acetyl, m/z 1274.0; malonyl, m/z 1318.0; succinyl, m/z 1332.0) and blue traces showed the ion intensities (10× magnified) for the mass of the deacylated peptide (m/z 1232.0). Black traces showed the ion intensity for all masses from 100-2000 (total ion counts or TIC). With Sirt1, hydrolysis was observed for the acetyl peptide (A), but not malonyl (B) or succinyl (C) peptide. With Sirt5, hydrolysis of the acetyl peptide was barely detectable (D) while hydrolysis of the malonyl (E) and succinyl (F) peptides were obvious.

The structure of Sirt5 revealed an unusual acyl pocket. (A) The acyl pocket of Sirt5 was partially occupied by the sulfate from the CHES molecule via interactions with Arg105 and Tyr102. The sulfur was 4.2 Å away from the thioacetyl group. (B) Alignment of Sirt5-thioacetyl peptide structure (grey) and Sir2Tm-acetyl peptide structure (PDB 2h4f, magenta). (C) The rationale for predicting that malonyl/succinyl peptides could be better substrates for Sirt5. (D) Sirt5-succinyl peptide-NAD ternary structure showing that the succinyl group interacted with Tyr102 and Arg105.

(A) Succinyl lysine was detected in bovine liver mitochondria. Sirt5-catalyzed hydrolysis of malonyl and succinyl peptides could be detected using ³²P-NAD, which formed ³²P-labeled O-Ma-ADPR (lane 2) and O-Su-ADPR (lane 3). No reaction occurred with acetyl peptide (lane 1). The formation of O-Ac-ADPR catalyzed by Sirt1 was detected (lanes 4 and 8). O-Su-ADPR was formed when bovine liver mitochondria peptides were incubated with Sirt5 (lane 9), but not with Sirt1 (lane 10). The control with BSA peptides and Sirt5 did not generate O-Su-ADPR (lane 12). CD38-catalzyed hydrolysis of NAD was used to generate the standard ³²P-ADPR spot (lane 13). (B) The CPS1 activities were measured using the liver lysates from Sirt5 wt and KO mice. (n=3, p<0.05). Relative quantitation analysis was achieved by extracted ion chromatograms (XICs) for peak areas of CPS1 K1291 acetyl peptides from Sirt5 KO (C) and wt mice (D); of CPS1 K1291 succinyl peptides from Sirt5 KO (E) and wt mice (F), and of a reference peptide from Sirt5 KO (G) and wt mice (H).