Side chain specificity of ADP-ribosylation by a sirtuin.
Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity, and cell proliferation, although few cellular ADP-ribosyltransferases have been identified. The sirtuins constitute a highly conserved family of protein deacetylases, and several family members have also been reported to perform protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog Trypanosoma brucei SIR2-related protein 1 (TbSIR2RP1) on both acetylated and unacetylated substrates. We demonstrated that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction performed by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and MS data showed that arginine is the major ADP-ribose acceptor for unacetylated substrates, whereas arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We performed combined ab initio quantum mechanical/molecular mechanical molecular dynamics simulations, which indicated that sirtuin ADP-ribosylation at arginine is energetically feasible, and involves a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in the deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can perform ADP-ribosylation using two distinct mechanisms, depending on whether or not the substrate is acetylated.
Fahie
Kamau
K
Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Hu
Po
P
Swatkoski
Stephen
S
Cotter
Robert J
RJ
Zhang
Yingkai
Y
Wolberger
Cynthia
C
D036002:ADP Ribose Transferases / Q000378:metabolism; D000107:Acetylation; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D008958:Models, Molecular; D015800:Protozoan Proteins / Q000378:metabolism; D037761:Sirtuins / Q000378:metabolism*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000378:metabolism
D015800:Protozoan Proteins; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D036002:ADP Ribose Transferases; D037761:Sirtuins