Biosynthesis of NAD(P) in bacteria occurs through either the de novo or one of the salvage pathways which converge at the point where the reaction of nicotinate mononucleotide (NaMN) with adenosine triphosphate (ATP), is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate (PP i ). This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT) which is essential for bacterial growth making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed to exhibit negative cooperativity, i.e. Hill coefficients < 1.0. Negative cooperativity in binding is also supported from X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN and NaAD-bound complexes were determined to resolutions of 2.50 Å, 2.60 Å and 1.75 Å, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model whereby the binding affinity of substrates to the first monomer of NMAT is stronger than to the second and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has so far not yet been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD (P) biosynthetic pathway may in part occur at the reaction catalyzed by NMAT.
The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts ( approximately 20,000 wells per day) to ultra-high throughput environments ( approximately 200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.
Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)-lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we describe the cloning and characterization of bovine eIF5A and bovine deoxyhypusine synthase. The deduced bovine eIF5A protein is 100% identical to human eIF5A-1, and the deduced bovine deoxyhypusine synthase protein showed a 93% identity to the human protein.
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