Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis

Suzanne F. O’Handley, Puchong Thirawatananond, Lin Woo Kang, Jennifer E. Cunningham, J. Alfonso Leyva, L. Mario Amzel, Sandra B. Gabelli

Research output: Research - peer-reviewArticle

Abstract

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+. Km and kcat 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 Km increased only slightly (2- to 3-fold) but the kcat 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.

LanguageEnglish (US)
Pages1-11
Number of pages11
JournalJournal of Bioenergetics and Biomembranes
DOIs
StateAccepted/In press - Sep 28 2016

Fingerprint

Adenosine Diphosphate Ribose
Hydrolases
Mycobacterium tuberculosis
Enzymes
Catalysis
Cats
Ions
Sequence Analysis
Oxidative Stress
Hydrolysis
Population
Genes
Proteins
Therapeutics
tellurous acid

Keywords

  • ADP-ribose
  • ADP-ribose hydrolase
  • ADPRase
  • Mycobacterium tuberculosis
  • Nudix

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis. / O’Handley, Suzanne F.; Thirawatananond, Puchong; Kang, Lin Woo; Cunningham, Jennifer E.; Leyva, J. Alfonso; Amzel, L. Mario; Gabelli, Sandra B.

In: Journal of Bioenergetics and Biomembranes, 28.09.2016, p. 1-11.

Research output: Research - peer-reviewArticle

O’Handley, Suzanne F. ; Thirawatananond, Puchong ; Kang, Lin Woo ; Cunningham, Jennifer E. ; Leyva, J. Alfonso ; Amzel, L. Mario ; Gabelli, Sandra B./ Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis. In: Journal of Bioenergetics and Biomembranes. 2016 ; pp. 1-11
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