Investigating the ADP-ribosyltransferase Activity of Sirtuins with NAD Analogues and ³²P-NAD

Abstract

Protein ADP-ribosyltransferases catalyze the transfer of adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) onto specific target proteins. Sirtuins, a class of enzymes with NAD-dependent deacetylase activity, have been reported to possess ADP-ribosyltransferase activity, too. Here we used NAD analogues and 32P-NAD to study the ADP-ribosyltransferase activity of several different sirtuins, including yeast Sir2, human SirT1, mouse SirT4, and mouse SirT6. The results showed that an alkyne-tagged NAD is the substrate for deacetylation reactions but cannot detect the ADP-ribosylation activity. Furthermore, comparing with a bacterial ADP-ribosyltransferase diphtheria toxin, the observed rate constant of sirtuin-dependent ADP-ribosylation is >5000-fold lower. Compared with the kcat/Km values of the deacetylation activity of sirtuins, the observed rate constant of sirtuin-dependent ADP-ribosyltion is 500 times weaker. The weak ADP-ribosylation events can be explained by both enzymatic and nonenzymatic reaction mechanisms. Combined with recent reports on several other sirtuins, we propose that the reported ADP-ribosyltransferase activity of sirtuins is likely some inefficient side reactions of the deacetylase activity and may not be physiologically relevant.

The Bicyclic Intermediate Structure Provides Insights into the Desuccinylation Mechanism of Human Sirtuin 5 (SIRT5)

“…Protein Cloning, Expression, and Purification-Truncated SIRT5 (34 -302) was cloned using TOPO and Gateway cloning technology (Invitrogen) into pDEST-F1 for expression, expressed in Escherichia coli, and purified as described previously (34). Purified protein was dialyzed into crystallization buffer (20 mM Tris pH 8.0, 20 mM NaCl, and 5% glycerol), concentrated to 16 mg/ml, flash-frozen in liquid nitrogen, and stored at Ϫ80°C for crystallization.…”

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

“…Rh-NAD was prepared as described previously (32). Purified yeast eEF-2 and Rh-NAD (25 μM) were incubated with DT at 30°C in 25 mM Tris·HCl (pH 8.0), 50 mM NaCl, 30 mM DTT, and 2 mM EDTA.…”

An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms

“…For Sirt4, no deacetylation substrate has yet been identified. Instead, Sirt4 was reported to regulate glutamate dehydrogenase through ADPribosylation 18 , an alternative, normally less efficiently catalysed reaction of sirtuins 19 . The major cytosolic isoform is Sirt2, which deacetylates, for example, a-tubulin 20 .…”