Sirtuins are NAD-dependent deacetylases that regulate important biological processes. Mammals have seven sirtuins, Sirt1-7. Four of them (Sirt4-7) have no detectable or very weak deacetylase activity. Here we found that Sirt5 is an efficient protein lysine desuccinylase and demalonylase in vitro. The preference for succinyl and malonyl groups was explained by the presence of an arginine residue (Arg105) and tyrosine residue (Tyr102) in the acyl pocket of Sirt5. Several mammalian proteins were identified to have succinyl or malonyl lysine modifications by mass spectrometry. Deletion of Sirt5 in mice appeared to increases the level of succinylation on carbamoyl phosphate synthase 1, a known target of Sirt5. Thus protein lysine succinylation may represent a posttranslational modification that can be reversed by Sirt5 in vivo.

The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

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SIRT1 and SIRT2 are deacetylase enzymes that belong to the sirtuin family and are involved in tumorigenesis. In our screen for small molecules inhibiting SIRT1/2 toxoflavin was identified. Toxoflavin potently inhibited SIRT1 activity in in vitro deacetylase assay using purified SIRT1 protein. SIRT2 activity was also inhibited by toxoflavin less potently than SIRT1 in deacetylase assay in vitro. Toxoflavin exhibited growth inhibition of various cancer cell lines including A549 lung cancer cells with a GI(50) of 48 nM. Toxoflavin treatment in A549 cells increased the acetylated form of p53, which is a substrate of SIRT1. The acetylation levels of α-tubulin, a SIRT2 substrate, were also increased by toxoflavin treatment dose-dependently. Several toxoflavin derivatives were synthesized to determine the preliminary structure-activity relationship of toxoflavin. Some of the toxoflavin derivatives showed highly selective inhibition against SIRT1. In conclusion, this study presented toxoflavin as a potent SIRT1/2 inhibitor with anticancer activity.