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Structures and mechanisms of Nudix hydrolases

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Abstract

Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contain the sequence motif or Nudix box, GX5EX7REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 109- to 1012-fold. The reactions are accelerated 103–105-fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes ⩾103–105-fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap4AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10–103-fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components.

Section snippets

Catalytic properties and biological role of MutT

The MutT pyrophosphohydrolase from Escherichia coli, a monomeric protein containing 129 residues [14], catalyzes the unusual hydrolysis of nucleoside- and deoxynucleoside-triphosphates (NTP) to yield the nucleotide (NMP) and inorganic pyrophosphate (PPi) Eq. (1).NTP4-+H2ONMP+PPi+H+

This prototypical member of the Nudix superfamily was the first to have its structure determined [3], [4] and its mechanism studied in detail [15], [16], [17]. The early mechanistic work on this enzyme has been

Catalytic properties of ADPRP

ADPRP from E. coli (Orf209) catalyzes the hydrolysis of ADP–ribose, with high specificity, by nucleophilic substitution by water at the adenosyl phosphorus, yielding AMP and the leaving group, ribose-5-P [33].ADPribose+H2OAMP+ribose-5-P+2H+

The catalytic power of this enzyme is 109.5 with ADP–ribose as substrate [34]. The biological role of ADPRP is to remove ADP–ribose which is cytotoxic in E. coli, since the accumulation of this metabolite somehow potentiates tellurite toxicity, and its free

Catalytic properties of Ap4AP

Ap4AP, a Nudix enzyme found in bacteria, plants, and animals, catalyzes the reaction:ApnA+H2OATP+Apn-3+2H+where n = 4, 5, or 6. Substitution always occurs on the δP, with ATP as the leaving group [43]. The biological role of Ap4AP may be to regulate the cellular levels of diadenosine signaling molecules. The presence of this enzyme in Bartonella bacilliformis correlates with the invasiveness of this organism into erythrocytes in the hemolytic disease, Oroya fever [44], [45]. The solution

Catalytic properties and biologic role of GDPMH

The Nudix enzyme, GDP-mannose mannosyl hydrolase (GDPMH) may participate in the regulation of bacterial cell wall biosynthesis and protein glycosylation by controlling the intracellular concentration of the glycosylating substrate, GDP-mannose [56]. GDPMH catalyzes the hydrolysis of GDP–sugars to form GDP and sugar [13].GDP-α-D-mannose+H2OGDP+β-D-mannose+H+

It is a highly atypical Nudix family member in that it contains a modified sequence motif (changes in bold), GX5EX7RUX2XEX2U, in which the

Messenger RNA-5′-decapping enzymes (hDcp2, Dcp2p)

Eukaryotic enzymes which hydrolytically decap the 5′ end of capped messenger RNA, cloned from various sources, including a human erythroleukemia cell line (hDcp2) [60], [61], and yeast (Dcp2p) [62], were found to contain the 23 residue Nudix sequence motif. These enzymes catalyze the decapping reaction:Me7Gp3-RNA+H2OMe7GDP+p-RNA+2H+which helps to control the rate of degradation of messenger RNA. The 420 residue human enzyme hDcp2 binds RNA, and contains a Nudix fold region between residues 98

Acknowledgments

We are grateful to Jenny P. Glusker and Stephen J. Lippard for valuable comments, and to Maurice J. Bessman for his helpful advice over the years.

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    This research was supported by National Institutes of Health Grants DK28616 (to A.S.M.) and GM066895 (to L.M.A.)

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