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 histone 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 alpha-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 beta-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.
French
Jarrod B
JB
Department of Pharmacology, Weill Cornell College of Medicine, 1300 York Avenue LC216, New York, New York 10065, USA.
Cen
Yana
Y
Sauve
Anthony A
AA
D000107:Acetylation; D000818:Animals; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000096:biosynthesis / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D037761:Sirtuins / Q000737:chemistry / Q000378:metabolism*
D006657:Histones; D010455:Peptides; D015800:Protozoan Proteins; D009243:NAD; C016949:N-epsilon-acetyllysine; D009244:NAD+ Nucleosidase; D037761:Sirtuins; D008239:Lysine