RESUMO
Mycobacterium tuberculosis represents one of the world's most devastating infectious agents - with one third of the world's population infected and 1.5 million people dying each year from this deadly pathogen. As part of an effort to identify targets for therapeutic intervention, we carried out the kinetic characterization of the product of gene rv1700 of M. tuberculosis. Based on its sequence and its structure, the protein had been tentatively identified as a pyrophosphohydrolase specific for adenosine diphosphate ribose (ADPR), a compound involved in various pathways including oxidative stress response and tellurite resistance. In this work we carry out a kinetic, mutational and structural investigation of the enzyme, which provides a full characterization of this Mt-ADPRase. Optimal catalytic rates were achieved at alkaline pH (7.5-8.5) with either 0.5-1 mM Mg2+ or 0.02-1 mM Mn2+. K m and k cat values for hydrolysis of ADPR with Mg2+ ions are 200 ± 19 µM and 14.4 ± 0.4 s-1, and with Mn2+ ions are 554 ± 64 µM and 28.9 ± 1.4 s-1. Four residues proposed to be important in the catalytic mechanism of the enzyme were individually mutated and the kinetics of the mutant enzymes were characterized. In the four cases, the K m increased only slightly (2- to 3-fold) but the k cat decreased significantly (300- to 1900-fold), confirming the participation of these residues in catalysis. An analysis of the sequence and structure conservation patterns in Nudix ADPRases permits an unambiguous identification of members of the family and provides insight into residues involved in catalysis and their participation in substrate recognition in the Mt-ADPRase.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Mycobacterium tuberculosis/enzimologia , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Hidrolases/metabolismo , Cinética , Mutação , Mycobacterium tuberculosis/genética , Pirofosfatases/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
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.