Transition State Structure and Inhibition of Rv0091, a 5′-Deoxyadenosine/5′-methylthioadenosine Nucleosidase from <i>Mycobacterium tuberculosis</i>

5′-Methylthioadenosine/<i>S</i>-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the <i>N</i>-ribosidic bond in 5′-methylthioadenosine (MTA) and <i>S</i>-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in <i>Mycobacterium tuberculosis</i>. Rv0091 was expressed in <i>Escherichia coli</i>, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5′-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1′-³H], [1′-¹⁴C], [5′-³H₂], [9-¹⁵N], and [7-¹⁵N]­MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted <i>via</i> quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1′–N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5′-hexylthio-DADMe-immucillinA) displaying a <i>K</i>_i value of 87 pM

Binding Isotope Effects for <i>para</i>-Aminobenzoic Acid with Dihydropteroate Synthase from <i>Staphylococcus aureus</i> and <i>Plasmodium falciparum</i>

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for <i>para</i>-aminobenzoic acid on the dihydropteroate synthase enzymes from <i>Staphylococcus aureus</i> and <i>Plasmodium falciparum</i> present distinct chemical environments. Specifically, we show that <i>para</i>-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from <i>P. falciparum</i> relative to that of <i>S. aureus</i>. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of <i>para</i>-aminobenzoic acid. The loop specific to <i>P. falciparum</i> is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site

Transition State Analogue Inhibitors of 5′-Deoxyadenosine/5′-Methylthioadenosine Nucleosidase from <i>Mycobacterium tuberculosis</i>

<i>Mycobacterium tuberculosis</i> 5′-deoxyadenosine/5′-methylthioadenosine nucleosidase (Rv0091) catalyzes the <i>N</i>-riboside hydrolysis of its substrates 5′-methylthioadenosine (MTA) and 5′-deoxyadenosine (5′-dAdo). 5′-dAdo is the preferred substrate, a product of radical <i>S</i>-adenosylmethionine-dependent enzyme reactions. Rv0091 is characterized by a ribocation-like transition state, with low N-ribosidic bond order, an N7-protonated adenine leaving group, and an activated but weakly bonded water nucleophile. DADMe-Immucillins incorporating 5′-substituents of the substrates 5′-dAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5′-Deoxy-DADMe-Immucillin-A was the most potent among the 5′-dAdo transition state analogues with a dissociation constant of 640 pM. Among the 5′-thio substituents, hexylthio-DADMe-Immucillin-A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state analogues differs from those of other bacterial homologues because of an altered hydrophobic tunnel accepting the 5′-substituents. Inhibitors of Rv0091 had weak cell growth effects on <i>M. tuberculosis</i> or <i>Mycobacterium smegmatis</i> but were lethal toward <i>Helicobacter pylori</i>, where the 5′-methylthioadenosine nucleosidase is essential in menaquinone biosynthesis. We propose that Rv0091 plays a role in 5′-deoxyadenosine recycling but is not essential for growth in these <i>Mycobacteria</i>