Biochemical characterization of recombinant nucleoside hydrolase from Mycobacterium tuberculosis H37Rv

Abstract

Tuberculosis (TB) is a major global health threat. There is a need for the development of more efficient drugs for the sterilization of the disease's causative agent, Mycobacterium tuberculosis (MTB). A more comprehensive understanding of the bacilli's nucleotide metabolic pathways could aid in the development of new anti-mycobacterial drugs. Here we describe expression and purification of recombinant iunH-encoded nucleoside hydrolase from MTB (MtIAGU-NH). Glutaraldehyde cross-linking results indicate that MtIAGU-NH predominates as a monomer, presenting varied oligomeric states depending upon binding of ligands. Steady-state kinetics results show that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Inosine and adenosine displayed positive homotropic cooperativity kinetics, whereas guanosine and uridine displayed hyperbolic saturation curves. Measurements of kinetics of ribose binding to MtIAGU-NH by fluorescence spectroscopy suggest two pre-existing forms of enzyme prior to ligand association. The intracellular concentrations of inosine, uridine, hypoxanthine, and uracil were determined and thermodynamic parameters estimated. Thermodynamic activation parameters (Ea, ΔG(#), ΔS(#), ΔH(#)) for MtIAGU-NH-catalyzed chemical reaction are presented. Results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiment, multiple sequence alignment, and molecular docking experiments are also presented. These data should contribute to our understanding of the biological role played by MtIAGU-NH.

The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis

“…40 However, guanosine also failed to rescue Mtb from GuaB2 inhibition or depletion toxicity even though this organism has both purine nucleoside phosphorylase 59 and nucleoside hydrolase activities. 60 One possible explanation for this is that Mtb is unable to transport guanosine, a conclusion also consistent with the observation that this organism lacks NCS1 and NCS2 nucleoside cation symporter family homologues. 37 Because GuaB2 is the only one of the three GuaB orthologues in Mtb with confirmed IMPDH activity, 24 our findings confirm that GuaB2 is the primary target of VCC234718 in Mtb .…”

Thermodynamics, functional and structural characterization of inosine–uridine nucleoside hydrolase from Leishmania braziliensis

“…16 The ITC data analysis for a-D-ribose binding to LbIU-NH yielded a value of 192 mM, which is lower than for C. fasciculata nucleoside hydrolase, 16 and lower than for M. tuberculosis nucleoside hydrolase (10 mM). 43 As no heat change could be detected upon binding of hypoxanthine (5 mM) to LbIU-NH, there appears to be no binding of this base to free enzyme. Product inhibition studies for C. fasciculata nucleoside hydrolase indicate weak hypoxanthine affinity for free enzyme.…”

“…However the nucleoside hydrolase from Mycobacterium tuberculosis has been shown to display positive cooperativity for inosine and adenosine. 43 Even though recombinant LbIU-NH catalyzed the hydrolysis of purine and pyrimidine nucleosides, fairly low activities were observed for adenosine, cytidine and guanosine (Table 1). These results indicate that L. braziliensis has apparently evolved to preferentially catalyze hydrolysis of inosine and, to a lesser extent, uridine.…”

EvaluatingaroAgene essentiality and EPSP synthase vulnerability inMycobacterium smegmatisunder different nutritional conditions

“…For each sample of gene KD in liquid medium, total protein was extracted in 0h and 18h, as previously described [25, 26]. Cellular pellets were washed twice with 10 mM Tris-HCl pH 8.0 and then collected by centrifugation (4,000 rpm, 15 min, 4°C) (Hitachi himac CR21G centrifuge) and resuspended in 2 mL of the same buffer.…”

“…The mouse was euthanized by deep isoflurane inhalation one month later, and blood was collected by the descendant aorta. Serum was separated by centrifugation at 10,000 x g for 10 min, aliquoted, and stored at −80°C [26]. The western blot was performed in triplicate.…”