Thermodynamic study of dihydrofolate reductase inhibitor selectivity

Sasso, Sophie; Gilli, Robert; Sari, J.C.; Rimet, Odile; Briand, Claudette

doi:10.1016/0167-4838(94)90053-1

Cited by 35 publications

(23 citation statements)

References 36 publications

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“…Trimethoprim is an antibiotic that is a million times more selective for bacterial DHFRs (E:TMP has K d = 6 pM) (7) over mammalian DHFRs (K d ∼1-10 mM for DHFRs from bovine liver, murine, and human) (7,22). All ecDHFRs containing the G51PEKN mutation bind TMP with K d values between 1 and 6 mM, which is comparable to values observed in mammalian DHFRs.…”

Section: Resultsmentioning

confidence: 73%

“…Because of its biological role, DHFR is an important therapeutic target for anticancer and antibacterial drugs (6). One of the inhibitors of DHFR is trimethoprim (TMP), which exhibits potent antibacterial properties because of its heavily biased selectivity toward bacterial DHFRs over mammalian DHFRs (6,7). Aside from trimethoprim inhibition, there are many catalytic/kinetic discrepancies between the human (8,9) and the Escherichia coli (5) enzymes.…”

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confidence: 99%

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Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Liu

Hanoian

French

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

141

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With the rapidly growing wealth of genomic data, experimental inquiries on the functional significance of important divergence sites in protein evolution are becoming more accessible. Here we trace the evolution of dihydrofolate reductase (DHFR) and identify multiple key divergence sites among 233 species between humans and bacteria. We connect these sites, experimentally and computationally, to changes in the enzyme's binding properties and catalytic efficiency. One of the identified evolutionarily important sites is the N23PP modification (∼mid-Devonian, 415-385 Mya), which alters the conformational states of the active site loop in Escherichia coli dihydrofolate reductase and negatively impacts catalysis. This enzyme activity was restored with the inclusion of an evolutionarily significant lid domain (G51PEKN in E. coli enzyme; ∼2.4 Gya). Guided by this evolutionary genomic analysis, we generated a human-like E. coli dihydrofolate reductase variant through three simple mutations despite only 26% sequence identity between native human and E. coli DHFRs. Molecular dynamics simulations indicate that the overall conformational motions of the protein within a common scaffold are retained throughout evolution, although subtle changes to the equilibrium conformational sampling altered the free energy barrier of the enzymatic reaction in some cases. The data presented here provide a glimpse into the evolutionary trajectory of functional DHFR through its protein sequence space that lead to the diverged binding and catalytic properties of the E. coli and human enzymes.phylogenetic | EVB

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Section: Resultsmentioning

confidence: 73%

mentioning

confidence: 99%

Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Liu

Hanoian

French

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

141

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“…This enzyme is critical in maintaining the intercellular pool of DHF, which is subsequently used in the biosynthesis of purine nucleotides and some amino acids. Given its biological importance, DHFR is an important drug target (2,3), and its function has been extensively studied (4)(5)(6)(7)(8)(9)(10)(11)(12). However, several fundamental mechanistic details remained incomplete.…”

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confidence: 99%

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

Liu

Francis

Layfield

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) has become a model for understanding enzyme catalysis, and yet several details of its mechanism are still unresolved. Specifically, the mechanism of the chemical step, the hydride transfer reaction, is not fully resolved. We found, unexpectedly, the presence of two reactive ternary complexes [enzyme:NADPH:7,8-dihydrofolate (E:NADPH:DHF)] separated by one ionization event. Furthermore, multiple kinetic isotope effect (KIE) studies revealed a stepwise mechanism in which protonation of the DHF precedes the hydride transfer from the nicotinamide cofactor (NADPH) for both reactive ternary complexes of the WT enzyme. This mechanism was supported by the pH-and temperature-independent intrinsic KIEs for the C-H→C hydride transfer between NADPH and the preprotonated DHF. Moreover, we showed that active site residues D27 and Y100 play a synergistic role in facilitating both the proton transfer and subsequent hydride transfer steps. Although D27 appears to have a greater effect on the overall rate of conversion of DHF to tetrahydrofolate, Y100 plays an important electrostatic role in modulating the pK a of the N5 of DHF to enable the preprotonation of DHF by an active site water molecule.kinetic isotope effect | mechanism | dihydrofolate reductase | synergism E scherichia coli dihydrofolate reductase (ecDHFR) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) through the transfer of a proR hydride from the C4 atom of NADPH to the C6 position of the dihydropterin ring of DHF (1). This enzyme is critical in maintaining the intercellular pool of DHF, which is subsequently used in the biosynthesis of purine nucleotides and some amino acids. Given its biological importance, DHFR is an important drug target (2, 3), and its function has been extensively studied (4-12). However, several fundamental mechanistic details remained incomplete. One unresolved issue is the chronological order of the DHF protonation and hydride transfer steps. Quantum mechanical/molecular mechanical (QM/MM) calculations favor a stepwise protonation-hydride transfer reaction mechanism (10, 13, 14), whereas recent experimental data were interpreted as a change in the reaction mechanism between a concerted process and a stepwise process as a function of pH (15).The nature of DHF N5 protonation is also not fully understood. D27 has been implicated in the protonation of the N5 position of NADPH (4,12,14,(16)(17)(18)) through a linked water molecule. However, it is not clear how the water molecule-promoted DHF protonation at the N5 of the pterin can change in response to protein conformation (19) and to different electrostatic environments. Recently, the Y100 residue, which is located only ca. 3.5 Å from both the amide of the nicotinamide and N8 of the pterin ring (Fig. 1), has been shown to play an important role in electrostatically facilitating the ecDHFR-catalyzed reaction (20, 21). The location of Y100 suggests that it may form hydrogen bonds with ...

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“…DHFR is a cytosolic enzyme that acquires a tightly folded conformation when bound to folate analogues like methotrexate (MTX) or the hydrophobic membrane-permeable derivative, trimetrexate (TMX) (8). DHFR, when occupied by these ligands, acquires resistance to proteinase K digestion, yielding a distinctive 21-kDa protease-resistant fragment.…”

mentioning

confidence: 99%

Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation

Tirosh¹,

Furman

Tortorella

et al. 2003

Journal of Biological Chemistry

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We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.

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Thermodynamic study of dihydrofolate reductase inhibitor selectivity

Cited by 35 publications

References 36 publications

Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation

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