ADP-ribosylation is a post-translational modification that occurs on chemically diverse amino acids, including aspartate, glutamate, lysine, arginine, serine and cysteine on proteins and is mediated by ADP-ribosyltransferases, including a subset commonly known as poly(ADP-ribose) polymerases. ADP-ribose can be conjugated to proteins singly as monomer or in polymeric chains as poly(ADP-ribose). While ADP-ribosylation can be reversed by ADP-ribosylhydrolases, this protein modification can also be processed to phosphoribosylation by enzymes possessing phosphodiesterase activity, such as snake venom phosphodiesterase, mammalian ectonucleotide pyrophosphatase/phosphodiesterase 1, Escherichia coli RppH, Legionella pneumophila Sde and Homo sapiens NudT16 (HsNudT16). Our studies here sought to utilize X-ray crystallographic structures of HsNudT16 in complex with monomeric and dimeric ADP-ribose in identifying the active site for binding and processing free and protein-conjugated ADP-ribose into phosphoribose forms. These structural data guide rational design of mutants that widen the active site to better accommodate protein-conjugated ADP-ribose. We identified that HsNudT16 Δ17, HsNudT16 F36A, and HsNudT16 F61S have lessened activity for free ADP-ribose, while have comparable processing ability against protein-conjugated mono(ADP-ribose). On the other hand, they improved catalytic efficiency for protein-conjugated poly(ADP-ribose). These HsNudT16 variants are a novel tool to investigate different forms of parylated proteins.

Protein engineering of Nudix hydrolases for processing of poly(ADP-ribosyl)ated proteins

2019 Fall National Meeting

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