Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosisH37Rv: an essential step in the function-based rational design of anti-TB drugs

Abstract

The number of new cases of tuberculosis (TB) arising each year is increasing globally. Migration, socio-economic deprivation, HIV co-infection and the emergence of drug-resistant strains of Mycobacterium tuberculosis, the main causative agent of TB in humans, have all contributed to the increasing number of TB cases worldwide. Proteins that are essential to the pathogen survival and absent in the host, such as enzymes of the shikimate pathway, are attractive targets to the development of new anti-TB drugs. Here we describe the metal requirement and kinetic mechanism determination of M. tuberculosis dehydroquinate synthase (MtDHQS). True steady-state kinetic parameters determination and ligand binding data suggested that the MtDHQS-catalyzed chemical reaction follows a rapid-equilibrium random mechanism. Treatment with EDTA abolished completely the activity of MtDHQS, and addition of Co(2+) and Zn(2+) led to, respectively, full and partial recovery of the enzyme activity. Excess Zn(2+) inhibited the MtDHQS activity, and isotitration microcalorimetry data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. We also report measurements of metal concentrations by inductively coupled plasma atomic emission spectrometry. The constants of the cyclic reduction and oxidation of NAD(+) and NADH, respectively, during the reaction of MtDHQS was monitored by a stopped-flow instrument, under single-turnover experimental conditions. These results provide a better understanding of the mode of action of MtDHQS that should be useful to guide the rational (function-based) design of inhibitors of this enzyme that can be further evaluated as anti-TB drugs.

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

“…The pET expression vector system has a strong IPTG-inducible bacteriophage T7 lacUV5 late promoter that controls the T7 RNA polymerase to transcribe cloned target genes [24]. However, lac -controlled systems could have high level protein expression in the absence of inducer due to derepression of the system when cells approach stationary phase in complex medium, as previously reported for other enzymes [19], [25]–[30].…”

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

“…The mycobacterial enzyme has the extraordinary ability to conduct six chemical reactions ( Figure 6) in a single active site, which was considered by some a marvel feat, attracting interest on the enzyme's structure and chemical mechanism [46][47][48]. The cloning and functional demonstration of MtDHQS activity was first reported by Mendonça and coworkers [49] and studies on its kinetic mechanism were subsequently performed [50]. Initial velocity measurements and fluorescence binding studies point to a rapid equilibrium ordered mechanism, with KM values of 6.3 μM for DAHP and 70 μM for NAD + .…”

“…DHQS is a metalloenzyme, requiring a divalent cation for its activity. Incubation with EDTA abolishes enzyme activity, which can be restored by addition of Co 2+ into the reaction mixture and by Zn 2+ , Ca 2+ , Cd 2+ , Mg 2+ , Mn 2+ , Ni 2+ and Ba 2+ to a smaller extent [50]. Although Cobalt seems to be more catalytically relevant, inductively plasma atomic absorption analysis of recombinant MtDHQS revealed the presence of Zinc in the enzyme preparation, probably due to the greater bioavailability of Zinc and in agreement with reports related to the DHQS domain of AROM from Aspergillus nidulans [51,52].…”

Toward a structome of Acinetobacter baumannii drug targets

“…25 3-Dehydroquinate synthase catalyzes the second step of the shikimate pathway facilitating the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate to dehydroquinate, requiring catalytic amounts of nicotinamide adenine dinucleotide (NAD+) and metal cofactors Co 2+ or Zn 2+ . 26,27 The shikimate pathway plays an essential role in bacteria, fungi, algae, and other microorganisms and is necessary for the biosynthesis of chorismic acid, a precursor to aromatic amino acids as well as other metabolites. 28 This pathway is an attractive target for drug development due to its absence in mammals and potential for low human toxicity.…”