Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography [Neutron and ultrahigh resolution X-ray crystallography reveals water as the proton donor in the catalytic mechanism of dihydrofolate reductase]

Wan, Qun; Bennett, Brad C.; Wilson, Mark A.; Kovalevsky, Andrey; Langan, Paul; Howell, Elizabeth E.; Dealwis, Chris

doi:10.1073/pnas.1415856111

Title: Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography [Neutron and ultrahigh resolution X-ray crystallography reveals water as the proton donor in the catalytic mechanism of dihydrofolate reductase]

Journal Article · Mon Dec 01 00:00:00 EST 2014 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1415856111· OSTI ID:1185785

Wan, Qun ^[1]; Bennett, Brad C. ^[2]; Wilson, Mark A. ^[3]; Kovalevsky, Andrey ^[4]; Langan, Paul ^[4]; Howell, Elizabeth E. ^[5]; Dealwis, Chris ^[6]

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou (People's Republic of China); Jiangsu Key Lab. of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Disease, Yangzhou (People's Republic of China); Yangzhou Univ. (People's Republic of China)
Univ. of Virginia, Charlottesville, VA (United States)
Univ. of Nebraska, Lincoln, NE (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Univ. of Tennessee, Knoxville, TN (United States)
Case Western Reserve Univ., Cleveland, OH (United States)

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to the lack of consensus on a catalytic mechanism. To resolve this ambiguity, we conducted neutron and ultrahigh resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of DHFR with folate and NADP⁺ from E. coli. The neutron data were collected to 2.0 Å resolution using a 3.6 mm³ crystal with the quasi-Laue technique, and the structure reveals that the N3 atom of folate is protonated while Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer due to protonation of the N3 atom, suggesting tautomerization is unnecessary for catalysis. In the 1.05 Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pK_a of the N5 atom of DHF by Asp27, and protonation of N5 by water whose access to the active site is gated by fluctuation of the Met20 side chain even though the Met-20 loop is closed.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). High Flux Isotope Reactor (HFIR)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1185785

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, Issue 51; ISSN 0027-8424

Publisher:: National Academy of Sciences, Washington, DC (United States)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 57 works

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