title: Transition State Structure and Inhibition of Rv0091, a 5'-Deoxyadenosine/5'-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis.
abstract: 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5'-methylthioadenosine (MTA) and S-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 Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, 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'-(3)H], [1'-(14)C], [5'-(3)H2], [9-(15)N], and [7-(15)N]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 via 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 Ki value of 87 pM.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Hilda A
authors.initials: HA
authors.lastname: Namanja-Magliano
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Stratton
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D003839:Deoxyadenosines / Q000737:chemistry; D050113:Imino Furanoses / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011744:Pyrimidinones / Q000737:chemistry*; D011759:Pyrrolidines / Q000737:chemistry*; D011789:Quantum Theory; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D013872:Thionucleosides / Q000737:chemistry
chemical_list: C000622580:5'-hexylthio-DADMe-immucillin A; D003839:Deoxyadenosines; D050113:Imino Furanoses; D011744:Pyrimidinones; D011759:Pyrrolidines; D013872:Thionucleosides; C033714:5'-deoxyadenosine; C008500:5'-methylthioadenosine; D012435:S-Adenosylhomocysteine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D000241:Adenosine