Sirt5 is an NAD-dependent protein lysine desuccinylase and demalonylase. The study shows that Sirt5 efficiently removes succinyl and malonyl groups from proteins in vitro. The preference for these groups is due to specific residues (Arg105 and Tyr102) in the acyl pocket of Sirt5. Mass spectrometry identified several mammalian proteins with succinyl or malonyl lysine modifications. Deletion of Sirt5 in mice increased succinylation of carbamoyl phosphate synthase 1, suggesting that protein lysine succinylation can be reversed by Sirt5 in vivo. Sirtuins are NAD-dependent deacetylases that regulate biological processes. Sirt5, unlike other sirtuins, lacks robust deacetylase activity. Structural analysis revealed that Sirt5's acyl pocket interacts with thioacetyl peptides, and the presence of CHES in the pocket explains its lower catalytic efficiency compared to Sirt1. The structure of Sirt5 in complex with a succinyl peptide and NAD showed that the succinyl group interacts with Tyr102 and Arg105, confirming their role in binding acyl groups. Sirt5 catalyzes the hydrolysis of malonyl and succinyl peptides, producing O-Ma-ADPR and O-Su-ADPR, which were detected by mass spectrometry. These products were not formed by Sirt1 with these peptides. Sirt5's desuccinylase and demalonylase activities are much higher than its deacetylase activity. The presence of Tyr102 and Arg105 in the active site explains the preference for negatively charged acyl groups. In vivo, Sirt5 was found to desuccinylate mitochondrial proteins, including carbamoyl phosphate synthase 1. Sirt5 knockout mice showed increased succinylation of CPS1, indicating that Sirt5 can reverse protein lysine succinylation. Sirt5 also catalyzes malonylation of proteins, as evidenced by the detection of malonyl and succinyl lysine in mitochondrial proteins. The study suggests that Sirt5 is an NAD-dependent demalonylase and desuccinylase, with higher activity for these modifications than deacetylation. The findings indicate that protein lysine succinylation and malonylation function similarly to acetylation, regulating metabolism in mammalian cells.Sirt5 is an NAD-dependent protein lysine desuccinylase and demalonylase. The study shows that Sirt5 efficiently removes succinyl and malonyl groups from proteins in vitro. The preference for these groups is due to specific residues (Arg105 and Tyr102) in the acyl pocket of Sirt5. Mass spectrometry identified several mammalian proteins with succinyl or malonyl lysine modifications. Deletion of Sirt5 in mice increased succinylation of carbamoyl phosphate synthase 1, suggesting that protein lysine succinylation can be reversed by Sirt5 in vivo. Sirtuins are NAD-dependent deacetylases that regulate biological processes. Sirt5, unlike other sirtuins, lacks robust deacetylase activity. Structural analysis revealed that Sirt5's acyl pocket interacts with thioacetyl peptides, and the presence of CHES in the pocket explains its lower catalytic efficiency compared to Sirt1. The structure of Sirt5 in complex with a succinyl peptide and NAD showed that the succinyl group interacts with Tyr102 and Arg105, confirming their role in binding acyl groups. Sirt5 catalyzes the hydrolysis of malonyl and succinyl peptides, producing O-Ma-ADPR and O-Su-ADPR, which were detected by mass spectrometry. These products were not formed by Sirt1 with these peptides. Sirt5's desuccinylase and demalonylase activities are much higher than its deacetylase activity. The presence of Tyr102 and Arg105 in the active site explains the preference for negatively charged acyl groups. In vivo, Sirt5 was found to desuccinylate mitochondrial proteins, including carbamoyl phosphate synthase 1. Sirt5 knockout mice showed increased succinylation of CPS1, indicating that Sirt5 can reverse protein lysine succinylation. Sirt5 also catalyzes malonylation of proteins, as evidenced by the detection of malonyl and succinyl lysine in mitochondrial proteins. The study suggests that Sirt5 is an NAD-dependent demalonylase and desuccinylase, with higher activity for these modifications than deacetylation. The findings indicate that protein lysine succinylation and malonylation function similarly to acetylation, regulating metabolism in mammalian cells.