NudT16

Introduction

NudT16 is a member of the Nudix superfamily of hydrolases which break a phosphoester bond between the two phosphates in nucleoside diphosphate-linked to moiety X proteins resulting in a nucleoside monophosphate (NMP) and a phosphate linked to moiety X. While NudT16 was initially described as a nuclear RNA and cytoplasmic mRNA decapping enzyme, further studies have shown that it also effectively hydrolyzes inosine diphsophate (IDP) and its hazardous deoxyribose cognate (dIDP) into inosine monophsophate (IMP) and deoxy inosine monophosphate (dIMP), respectively [1]. NudT16 has also been shown to regulate levels of 53BP1, an adaptor protein that recruits other proteins to the site of a DNA breakage, through hydrolytic removal of ADP-ribose from ADP-ribosylated 53BP1 [2].

Structure

NudT16 is a homodimer, consisting of two monomers of the same sequence. A structure on the left shows one monomer in cyan while the other in purple. This dimerization allows for each subunit to have deeper . Each monomer consists of two beta sheets surrounded by alpha-helices, as is typical of Nudix hydrolases. A variation of the catalytically relevant Nudix box characteristic of Nudix hydrolases consisting of 23 highly conserved residues (G₁Z_2-6E₇Z_8-14R₁₅E₁₆U₁₇Z₁₈E₁₉E₂₀Z₂₁G₂₂U₂₃ where Z is any amino acid and U is an aliphatic and hydrophobic residue) is present in NudT16 (GARRLELGEALALGSGWRHVCHA). In contrast to the negatively charged pockets lined with glutamate residues where metal ligands bind, the adenosine binding pocket is positively charged. The mouth of the binding site is about 9Å in width. Thirawatananond et. al. investigated whether widening of this mouth would allow proteins conjugated to ADP further into the binding site. Contrary to Nudix ADPRases, HsNudT16 binds adenosine of ADPr and buries it deep in the core, while leaving the non-adenosine ribose exposed to the surface. This orientation allows the exposed ribose to conjugate another protein[3]. Many in the mouth of this binding pocket are also involved in hydrogen bonding, the binding of metal ligands, and also serve to delimit the binding site.

Function

HsNudT16 has a variety of functions including hydrolysis of certain biomolecules and keeping the cell free of harmful nucleotides. It is responsible for hydrolyzing inosine triphosphate or diphosphate. This protein also has a large substrate specificity and has mRNA decapping capability. Thirawatananond et. al. investigated whether the widening of the ADPr binding site would allow for increased hydrolysis activity by NudT16. The group designed such mutants (F36A, F61S, and a double mutant with both F36A and F61S) and found that for these mutants, NudT16 hydrolysis activity decreased in free ADPr, remained comparably efficient in mono(ADP-ribosylated) proteins, and increased in poly(ADP-ribosylated) proteins. ^[1] NudT16 is a (Deoxy)inosine diphosphatase. Iyama et. al. found that HsNudT16 binds strongly to GTP, ITP, and XTP. The loss of this protein is followed by increased accumulation of single-strand breaks in DNA, reduced proliferation, and increased cell arrest. They also found increased levels of inosine in RNA, which informed the conclusion that HsNudT16 functions in the nucleus to protect the cell from ITP and its detrimental effects. ^[2]