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Insights into the Catalytic Mechanism and Transition State Stabilization of 5′‐Methylthioadenosine Nucleosidase using Neutron Crystallography
Author(s) -
Banco Michael T.,
Mishra Vidhi,
Ostermann Andreas,
Schrader Tobias E.,
Evans Gary B.,
Kovalevsky Andrey Y.,
Ronning Donald R.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.913.3
Subject(s) - chemistry , protonation , hydrogen bond , crystallography , stereochemistry , catalysis , oxocarbenium , crystal structure , carboxylate , molecule , nucleophile , organic chemistry , ion
5′‐Methylthioadenosine nucleosidase (MTAN) is an obligatory homodimeric protein that catalyzes the hydrolysis of the N ‐ribosidic bond of a variety of adenosine‐containing metabolites. The proposed catalytic mechanism of MTAN is initiated by D198 protonating the N7 position of the adenyl moiety, which consequently causes elongation and ultimate breaking of the N ‐ribosidic bond, resulting in the formation of an oxocarbenium ion intermediate. Of particular interest is the p K a elevation of D198 that was proposed to have a value of 8.2, which is significantly higher than the theoretical p K a for an aspartic acid side chain. In this study we present the first joint X‐ray/neutron crystal structures of Helicobacter pylori MTAN (HpMTAN) in complex with a substrate ( S ‐adenosylhomocysteine), early (Formycin A) and late ( p ‐ClPh‐Thio‐DADMe‐ImmA) dissociative transition state analogues, as well as with the catalytic products (adenine and S ‐ribosylhomocysteine). The solved X‐ray/neutron crystal structures provide the positions of exchangeable hydrogen atoms and protonation states of ionizable groups that further our understanding of the catalytic mechanism as well as the stabilization of the transition state for HpMTAN. These results confirm that the catalytic reaction is initiated by D198 functioning as a general acid through the observation of a shared deuterium atom with unconventional hydrogen bond geometry between the carboxylate group of D198 and the N7 position of adenine. Additionally, the transition state analogues neutron structures illustrate the minor differences in the hydrogen bond interactions resulting in the DADMe‐ImmA analogues being extremely potent inhibitors with K d values in the picomolar range, despite HpMTAN shown to contain an early dissociative transition state structure. Support or Funding Information 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.).