Identification of a key water molecule involved in the macrophage migration inhibitory factor-catalyzed tautomerization of para-hydroxyphenylpyruvate using neutron crystallography.

Authors

Gabriela C Schröder, Gregg V Crichlow, Ewelina Jablonowski, Georgios Pantouris, Jay Nix, Naomi Chayen, Flora Meilleur, Elias J Lolis

Published in

Protein science : a publication of the Protein Society. Volume 35. Issue 9. Pages e70666.

Abstract

Neutron crystallography was used to determine a 2.5-Å resolution all-atom structure of macrophage migration inhibitory factor (MIF) interacting with 3-(4-hydroxyphenyl)-pyruvate (HPP). MIF is a pro-inflammatory, pro-tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF-catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction-derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine-32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N-terminal Pro-1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room-temperature synchrotron x-ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation-induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.

PMID:
42568349
Bibliographic data and abstract were imported from PubMed on 08 Aug 2026.

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