Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase

Sirtuins are NAD(+)-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyl transfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyl transfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of historic peptide sequences. Interestingly, the enzyme can also hydrolyze NAD(+). Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces (x-stereochemistry as established by reaction of methanol which forms alpha-1-O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate P-stereochemistry. Stereochemistry was established by isolation of beta-1-O-methyl-ADPR when methanol was added as a cosolvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin-catalyzed ADP-ribosyl transfer.