Structural Basis for Different Substrate Specificities of Two ADP-Ribose Pyrophosphatases from Thermus thermophilus HB8

Abstract

ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins ofThermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg 2؉ , with Mg 2؉ and AMP, and with Mg 2؉ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg 2؉ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.Nudix proteins catalyze the hydrolysis of nucleoside diphosphate (NDP) linked to another moiety, x (e.g., nucleoside triphosphate [NTP], diadenosine polyphosphate [Ap n A, where n ϭ 2 to 6], NDP-sugar, NADH, and coenzyme A [CoA], etc). Nudix proteins are widely distributed in nature, including in viruses, bacteria, archaea, and eukaryotes, and share a highly conserved amino acid sequence motif called a "Nudix box" (GX 5 EX 7 RE7UXEEXGU, where U represents I, L, or V and X represents any amino acid), which forms a loop-helix-loop structure involved in catalysis (3,7,17). It has been proposed that the physiological function of Nudix proteins is "housecleaning" to eliminate potentially toxic nucleotide metabolites from cells and to regulate the concentrations of NDP derivatives. However, the catalytic mechanism, including substrate recognition and physiological function, remains to be elucidated for many Nudix proteins.ADP-ribose (ADPR) is one of the main substrates of Nudix proteins in all three kingdoms. ADPR is produced enzymatically as part of the turnover of NAD, cyclic ADPR, ADPribosylated proteins, and poly-ADP-ribosylated proteins (37). High intracellular levels of ADPR could result in nonenzymatic ADP ribosylation and interfere with the recognition of enzymatic ADP ribosylation (11). It is likely that ADPR pyrophosphatase (ADPRase) functions to eliminate potentially deleterious ADPR. ADPRases are distributed widely throughout the three kingdoms, and many organisms have two or more ADPRases (6, 22-24, 27, 33). The specificity for ADPR over other substrates varies among the ...

Molecular Mechanism of ADP-Ribose Hydrolysis By Human NUDT5 From Structural and Kinetic Studies

“…14,15,17,18 It has been shown that for EcADPRase, loop L9 undergoes significant conformational changes during catalysis and Glu162 on loop L9 acts as the general base to deprotonate a water molecule that further functions as the attacking nucleophile. 14 As the structural comparison of the hNUDT5-AMPCPR-Mg 2 + complex and the EcADPRase-AMPCPR-Mg 2 + complex shows that Asp164 of hNUTD5 occupies a similar position as that of Glu162 of EcADPRase, Asp164 was thought to be the equivalent residue to function as a general base.…”

“…13,15 However, a different catalytic mechanism was proposed for Thermus thermophilus ADPRase (TtADPRase) Ndx4 because only two metal ions are found to bind at the active site and thus are suggested to be involved in activation of a conserved water molecule that acts as the nucleophile to attack the α-phosphate of the substrate, and mutation of Glu127, which is considered to be equivalent to Glu162 of EcADPRase, has no effect on the enzymatic activity. [16][17][18] Intriguingly, in the structure of another TtADPRase Ndx2, there are three metal ions bound at the active site and mutation of Glu136 (corresponding to Glu162 of EcADPRase) does decrease k cat by about 12-fold. 18 For eukaryotes, crystal structures of two human ADPRases (hNUDT9 and hNUDT5) have been reported.…”

“…[16][17][18] Intriguingly, in the structure of another TtADPRase Ndx2, there are three metal ions bound at the active site and mutation of Glu136 (corresponding to Glu162 of EcADPRase) does decrease k cat by about 12-fold. 18 For eukaryotes, crystal structures of two human ADPRases (hNUDT9 and hNUDT5) have been reported. 19,20 The two enzymes belong to different groups and show distinct characteristics: hNUDT9 is active as a monomer and is highly specific for ADPR, 19,21 whereas hNUDT5 exists as a homodimer and can utilize a variety of ADP-sugar conjugates as the substrate, with a preference for ADPR.…”

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

“…Assay for cAMP and cGMP Phosphodiesterases-To determine the phosphodiesterase steady-state kinetic parameters, the following reaction mixture (80 l) was applied to a CAPCELL PAK C18 column as previously described (42). The reaction mixture (100 l), which contained 50 mM Tris-HCl, 100 mM KCl, 5 mM MnCl 2 , 0.1-2 mM cAMP or cGMP, and 1 M TTHA0118 (pH 7.5), was incubated at 37°C.…”

“…Assay for pAp Phosphatase Activity-To determine the pAp phosphatase steady-state kinetic parameters, hydrolytic activity was analyzed by measuring the production of inorganic orthophosphate using a colorimetric assay (42). The reaction mixture (100 l), which contained 50 mM Tris-HCl, 100 mM KCl, 5 mM MnCl 2 or MgCl 2 , 5-1,000 M pAp, and 0.2 nM TTHA0118 or Mpn140 (pH 7.5), was incubated at 37°C.…”