Structure and Mechanism of MT-ADPRase, a Nudix Hydrolase from Mycobacterium tuberculosis

Abstract

Nudix hydrolases are a family of proteins that contain the characteristic sequence GX(5)EX(7)REUXEEXG(I/L/V), the Nudix box. They catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives such as ADP-ribose, Ap(n)A (3 </= n </= 6), NADH, and dATP. A number of Nudix hydrolases from several species, ranging from bacteria to humans, have been characterized, including, in some cases, the determination of their three-dimensional structures. The product of the Rv1700 gene of M. tuberculosis is a Nudix hydrolase specific for ADP-ribose (ADPR). We have determined the crystal structures of MT-ADPRase alone, and in complex with substrate, with substrate and the nonactivating metal ion Gd(3+), and in complex with a nonhydrolyzable ADPR analog and the activating metal ion Mn(2+). These structures, refined with data extending to resolutions between 2.0 and 2.3 A, showed that there are sequence differences in binding site residues between MT-ADPRase and a human homolog that may be exploited for antituberculosis drug development.

Structural Insights into the Thermus thermophilus ADP-ribose Pyrophosphatase Mechanism via Crystal Structures with the Bound Substrate and Metal

“…Smaller loops, such as the loop-helix structure described above, are advantageous to TtADPRase. The TtADPRase structure of Gd 3ϩ ternary complex was quite similar to those of the EcADPRase and MtADPRase (10,11), although the detailed structures differed. N-terminal domain ␤-sheet was shorter, loop L4 was moved outward, and loop L5 tip was situated upward by 4 Å. TtADPRase has other properties that might contribute to stability, including a lower content of chemically unstable amino acids such as Asn, Cys, and Met (25), and a smaller surface area in the N-terminal domain than for EcADPRase and MtADPRase.…”

“…According to the EcADPRase and MtADPRase structures of ternary complex with substrate analogue and metal ions, EcGlu-162 and MtGlu-142 in loop L9 are believed to act as catalytic bases (10,11). When these ternary complex structures were superimposed on that of TtADPRase, the results showed that loop L4 of TtADPRase corresponds to loop L9 of EcADPRase and MtADPRase.…”

“…The structures of the free enzyme, the Zn 2ϩ -bound enzyme, the binary complex with ADPR, the ternary complexes with ADPR and metal, and the product complexes containing AMP or ribose 5Ј-phosphate were determined. Based on structural and functional analyses, we propose a novel reaction mechanism for ADPR pyrophosphate hydrolysis that is different from those proposed based on the ternary complex structures of ADPRases from Escherichia coli (EcADPRase) and from Mycobacterium tuberculosis (MtADPRase) (10,11).…”

Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

“…In IPAV, the ADPRase (LIF_A0876) has a point mutation in the conserved amino-acid residue 148 that leads to a substitution of acidic Asp by nonpolar Gly in the C-terminal domain. According to the structural study of the ADPRase of Mycobacterium tuberculosis (its corresponding amino acid is also an acidic residue), this site forms a conserved loop that contacts the substrate, triggers a conformation change of the enzyme and finally accomplishes catalysis [57]. Therefore, the alteration in IPAV ADPRase is likely to be deleterious, pending experimental confirmation.…”

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

“…The Nudix superfamily exhibits a great deal of functional diversity. In addition to helping control the concentrations of crucial metabolites (e.g., ADP-ribose [12,19], UDP-glucose [46], and coenzyme A [4]), members of the superfamily also play key roles in biosynthetic pathways (e.g., folate biosynthesis [13]). It has been shown that some members of the Nudix family, i.e., Schizosaccharomyces pombe Dcp2 (31) and Escherichia coli and Bdellovibrio bacteriovorax RppH (7,23), may also play a role in RNA degradation by decapping mRNA.…”