Abstract
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-deazaadenin-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.
Original language | English |
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Pages (from-to) | 13756-13761 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 113 |
Issue number | 48 |
DOIs | |
State | Published - Nov 29 2016 |
Funding
We thank Heinz Maier-Leibnitz Zentrum and Oak Ridge National Laboratory for graciously providing the beam time that was essential for this work. This work was supported by the Center for the Advancement of Science in Space via a cooperative agreement with National Aeronautics and Space Administration Grant N-123528-01 (to D.R.R) and by National Institute of Allergy and Infectious Disease/NIH Grant AI105084 (to D.R.R.). This research used the resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and by Grant 085P1000817 from the Michigan Technology Tri-Corridor. The research was sponsored in part by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The IMAGINE Project was partially supported by the National Science Foundation (Grant 0922719).
Funders | Funder number |
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Heinz Maier-Leibnitz Zentrum | |
Scientific User Facilities Division | |
National Science Foundation | 0922719 |
National Institutes of Health | |
U.S. Department of Energy | |
National Institute of Allergy and Infectious Diseases | R01AI105084 |
National Aeronautics and Space Administration | N-123528-01 |
Michigan Economic Development Corporation | 085P1000817 |
Office of Science | |
Basic Energy Sciences | |
Argonne National Laboratory | DE-AC02-06CH11357 |
Oak Ridge National Laboratory | |
Michigan Technology Tri-Corridor |
Keywords
- Enzyme mechanism
- Helicobacter
- Neutron diffraction
- Nucleosidase
- Proton transfer