Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase

Jiayue Li; Jennifer Lin; Amnon Kohen; Priyanka Singh; Kevin Francis; Christopher M Cheatum

doi:10.1021/acs.biochem.1c00558

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Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase

Journal article

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Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase

Jiayue Li, Jennifer Lin, Amnon Kohen, Priyanka Singh, Kevin Francis and Christopher M Cheatum

Biochemistry (Easton), Vol.60(50), pp.3822-3828

12/21/2021

DOI: 10.1021/acs.biochem.1c00558

PMCID: PMC8697555

PMID: 34875176

Appears in UI Libraries Support Open Access

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Abstract

Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from DHFR (ecDHFR) as a model, but less studies have used the enzyme from DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction. The corresponding sites in hsDHFR are F28, 62-PEKN, and 26-PPLR. Each of these sites was studied here through the creation of mutant variants of the enzyme and measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs) on the reaction. F28 is mutated first to M (F28M) and then to the L of the bacterial enzyme (F28L). The KIEs of the F28M variant are larger and more temperature-dependent than wild-type (WT), suggesting a broader and longer average DAD at the TRS. To more fully mimic ecDHFR, we also study a triple mutant of the human enzyme (F32L-PP26N-PEKN62G). Remarkably, the intrinsic KIEs, while larger in magnitude, are temperature-independent like the WT enzymes. We also construct deletion mutations of hsDHFR removing both the 62-PEKN and 26-PPLR sequences. The results mirror those described previously for insertion mutants of ecDHFR. Taken together, these results suggest a balancing act during DHFR evolution between achieving an optimal TRS for hydride transfer and preventing product inhibition arising from the different intercellular pools of NADPH and NADP in prokaryotic and eukaryotic cells.

Thermodynamics

Amino Acid Substitution

Biocatalysis

Escherichia coli - enzymology

Escherichia coli - genetics

Evolution, Molecular

Humans

Kinetics

Models, Molecular

Mutagenesis, Site-Directed

Protein Conformation

Tetrahydrofolate Dehydrogenase - chemistry

Tetrahydrofolate Dehydrogenase - genetics

Tetrahydrofolate Dehydrogenase - metabolism

UIOWA OA Agreement

Details

Title: Subtitle: Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase
Creators: Jiayue Li - Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
Jennifer Lin - Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
Amnon Kohen - Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
Priyanka Singh - Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
Kevin Francis - Texas A&M University-Kingsville, Kingsville, Texas 78363, United States
Christopher M Cheatum - Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.60(50), pp.3822-3828
DOI: 10.1021/acs.biochem.1c00558
PMID: 34875176
PMCID: PMC8697555
NLM abbreviation: Biochemistry
ISSN: 0006-2960
eISSN: 1520-4995
Publisher: American Chemical Society
Grant note: R01 GM065368 / NIGMS NIH HHS
Language: English
Date published: 12/21/2021
Academic Unit: Liberal Arts and Science Admin; Chemistry
Record Identifier: 9984216711602771

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