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标题：Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states
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作者：Michael T. Banco ; Vidhi Mishra ; Andreas Ostermann 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：48
页码：13756-13761
DOI：10.1073/pnas.1609718113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceGastrointestinal infection by the bacterium Helicobacter pylori is strongly associated with the development of gastric cancer. H. pylori 5'-methylthioadenosine nucleosidase (HpMTAN) is an interesting drug target because of its vital role in the production of menaquinone. HpMTAN offers a unique target for treating H. pylori infections without affecting the survival of the human microbiome. Neutron crystallography was performed to determine hydrogen atom positions that provide insight into the catalytic mechanism and transition state stabilization. MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.
关键词：neutron diffraction ; enzyme mechanism ; proton transfer ; nucleosidase ; Helicobacter