Substrate specificity is critically important for enzyme catalysis. In the adrenaline-synthesizing enzyme PNMT (phenylethanolamine N-methyltransferase), minor changes in substituents can convert substrates into inhibitors. Here we report the crystal structures of six human PNMT complexes, including the first structure of the enzyme in complex with its physiological ligand R-noradrenaline. Determining this structure required rapid soak methods because of the tendency for noradrenaline to oxidize. Comparison of the PNMT–noradrenaline complex with the previously determined PNMT–p-octopamine complex demonstrates that these two substrates form almost equivalent interactions with the enzyme and show that p-octopamine is a valid model substrate for PNMT. The crystal structures illustrate the adaptability of the PNMT substrate binding site in accepting multi-fused ring systems, such as substituted norbornene, as well as noradrenochrome, the oxidation product of noradrenaline. These results explain why only a subset of ligands recognized by PNMT are methylated by the enzyme; bulky substituents dictate the binding orientation of the ligand and can thereby place the acceptor amine too far from the donor methyl group for methylation to occur. We also show how the critical Glu185 catalytic residue can be replaced by aspartic acid with a loss of only 10-fold in catalytic efficiency. This is because protein backbone movements place the Asp185 carboxylate almost coincident with the carboxylate of Glu185. Conversely, replacement of Glu185 by glutamine reduces catalytic efficiency almost 300-fold, not only because of the loss of charge, but also because the variant residue does not adopt the same conformation as Glu185.
Skip Nav Destination
Article navigation
September 2009
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
August 27 2009
Molecular recognition of physiological substrate noradrenaline by the adrenaline-synthesizing enzyme PNMT and factors influencing its methyltransferase activity
Nyssa Drinkwater;
Nyssa Drinkwater
*Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia
Search for other works by this author on:
Christine L. Gee;
Christine L. Gee
2
*Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia
Search for other works by this author on:
Munish Puri;
Munish Puri
1
*Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia
Search for other works by this author on:
Kevin R. Criscione;
Kevin R. Criscione
†Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045-7582, U.S.A.
Search for other works by this author on:
Michael J. McLeish;
Michael J. McLeish
‡Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, U.S.A.
Search for other works by this author on:
Gary L. Grunewald;
Gary L. Grunewald
†Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045-7582, U.S.A.
Search for other works by this author on:
Jennifer L. Martin
Jennifer L. Martin
3
*Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia
3To whom correspondence should be addressed (email [email protected]).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
May 06 2009
Revision Received:
June 23 2009
Accepted:
July 02 2009
Accepted Manuscript online:
July 02 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 422 (3): 463–471.
Article history
Received:
May 06 2009
Revision Received:
June 23 2009
Accepted:
July 02 2009
Accepted Manuscript online:
July 02 2009
Citation
Nyssa Drinkwater, Christine L. Gee, Munish Puri, Kevin R. Criscione, Michael J. McLeish, Gary L. Grunewald, Jennifer L. Martin; Molecular recognition of physiological substrate noradrenaline by the adrenaline-synthesizing enzyme PNMT and factors influencing its methyltransferase activity. Biochem J 15 September 2009; 422 (3): 463–471. doi: https://doi.org/10.1042/BJ20090702
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.