Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis.
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+. Kmand kcatvalues 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 Kmincreased only slightly (2- to 3-fold) but the kcatdecreased 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.
School of Chemistry and Materials Science, Rochester Institute of Technology, Rochester, NY, 14623, USA.
Suzanne F
SF
O'Handley
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Puchong
P
Thirawatananond
Department of Biological Sciences, Konkuk University, Seoul, South Korea.
Lin-Woo
LW
Kang
School of Chemistry and Materials Science, Rochester Institute of Technology, Rochester, NY, 14623, USA.
Jennifer E
JE
Cunningham
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.|Physics Department, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
J Alfonso
JA
Leyva
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
L Mario
LM
Amzel
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. gabelli@jhmi.edu.|Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. gabelli@jhmi.edu.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. gabelli@jhmi.edu.
Sandra B
SB
Gabelli
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000246:Adenosine Diphosphate Ribose; D006867:Hydrolases; D011755:Pyrophosphatases; C019620:ADPribose pyrophosphatase
ADP-ribose; ADP-ribose hydrolase; ADPRase; Mycobacterium tuberculosis; Nudix
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