Namanja-Magliano Hilda A, Stratton Christopher F, Schramm Vern L
Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
ACS Chem Biol. 2016 Jun 17;11(6):1669-76. doi: 10.1021/acschembio.6b00144. Epub 2016 Apr 8.
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.
5'-甲硫基腺苷/S-腺苷高半胱氨酸核苷酶（MTAN）是一种细菌酶，可催化5'-甲硫基腺苷（MTA）和S-腺苷高半胱氨酸（SAH）中N-核糖苷键的水解。MTAN活性与群体感应途径、多胺生物合成和腺嘌呤补救有关。此前，Rv0091的编码序列在结核分枝杆菌中被注释为假定的MTAN。Rv0091在大肠杆菌中表达，纯化至同质，并显示为同二聚体，这与来自其他微生物的MTAN一致。Rv0091的底物特异性显示，相对于MTA或SAH，它更倾向于5'-脱氧腺苷。测定了[1'-(3)H]、[1'-(14)C]、[5'-(3)H2]、[9-(15)N]和[7-(15)N]MTA水解的本征动力学同位素效应（KIEs）分别为1.207、1.038、0.998、1.021和0.998。通过将量子化学计算预测的KIE值与本征KIEs相匹配，确定了Rv0091过渡态结构的模型。过渡态显示C1'-N9键级大幅降低，核糖环具有发达的氧碳鎓特征，水亲核试剂的参与较弱。Rv0091过渡态结构的静电势表面图显示与DADMe-亚胺青霉素过渡态类似物相似。DADMe-亚胺青霉素过渡态类似物对Rv0091表现出强烈抑制作用，最有效的抑制剂（5'-己硫基-DADMe-亚胺青霉素A）的Ki值为87 pM。
