Production of Dihydrofolate Reductase by Cloned<i>Escherichia coli</i>and Its Application to Asymmetric Synthesis of<i>l</i>-Leucovorin

Ohshiro, Takashi; Kuge, Yukihiro; Igarashi, Ayuko; Mochida, Kenichi; Iwakura, Masanori; Uwajima, Takayuki

doi:10.1271/bbb.56.437

Cited by 5 publications

(4 citation statements)

References 6 publications

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“…Recently, this strategy was applied to a glycoside hydrolase and HIV-1 protease, where neutron structures across a range of pH enabled the observation of isolated protons in the active site and allowed the pK a determination of catalytic acidic residues. 6,7 In this study, neutron diffraction experiments were conducted on the ecDHFR−folate−NADP + complex at pH 4.5, which is ∼2 pH units below the pH optimum of the ecDHFR 38 and well below the pH (7.0) of our previously determined pseudo-Michaelis structure. This acidic pH results in an ∼300-fold elevated concentration of free protons, increasing the probability of visualizing the elusive proton postulated to protonate the N5 atom of DHF.…”

Section: ■ Introductionmentioning

confidence: 99%

“…In this study, neutron diffraction experiments were conducted on the ecDHFR–folate–NADP + complex at pH 4.5, which is ∼2 pH units below the pH optimum of the ecDHFR 38 and well below the pH (7.0) of our previously determined pseudo-Michaelis structure. This acidic pH results in an ∼300-fold elevated concentration of free protons, increasing the probability of visualizing the elusive proton postulated to protonate the N5 atom of DHF.…”

Section: Introductionmentioning

confidence: 99%

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Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid–Base Catalysis

et al. 2021

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Acid–base catalysis, which involves one or more proton transfer reactions, is a chemical mechanism commonly employed by many enzymes. The molecular basis for catalysis is often derived from structures determined at the optimal pH for enzyme activity. However, direct observation of protons from experimental structures is quite difficult; thus, a complete mechanistic description for most enzymes remains lacking. Dihydrofolate reductase (DHFR) exemplifies general acid–base catalysis, requiring hydride transfer and protonation of its substrate, DHF, to form the product, tetrahydrofolate (THF). Previous X-ray and neutron crystal structures coupled with theoretical calculations have proposed that solvent mediates the protonation step. However, visualization of a proton transfer has been elusive. Based on a 2.1 Å resolution neutron structure of a pseudo-Michaelis complex of E. coli DHFR determined at acidic pH, we report the direct observation of the catalytic proton and its parent solvent molecule. Comparison of X-ray and neutron structures elucidated at acidic and neutral pH reveals dampened dynamics at acidic pH, even for the regulatory Met20 loop. Guided by the structures and calculations, we propose a mechanism where dynamics are crucial for solvent entry and protonation of substrate. This mechanism invokes the release of a sole proton from a hydronium (H 3 O + ) ion, its pathway through a narrow channel that sterically hinders the passage of water, and the ultimate protonation of DHF at the N5 atom.

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Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid–Base Catalysis

et al. 2021

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“…1.3) is a monomeric, two-domain protein with 159 amino acids that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as the reducing cofactor 17 , 18 . DHFR is a clinically important enzyme: it is the target of a number of antifolate drugs, such as trimethoprim (TMP) and methotrexate (MTX), and is involved in the production of an anticancer drug, 1-leucovorin 19 . DHFR is also a widely utilized model protein in the study of folding.…”

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Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase

et al. 2011

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Decoding sequence information is equivalent to elucidating the design principles of proteins. For this purpose, we conducted systematic alanine insertion analysis to reveal the regions in the primary structure where the sequence continuity cannot be disrupted. We applied this method to dihydrofolate reductase (DHFR), and examined the effects of alanine insertion on structure and the enzymatic activity by solubility assay and trimethoprim resistance, respectively. We revealed that DHFR is composed of “Structure Elements”, “Function Elements” and linkers connecting these elements. The “Elements” are defined as regions where the alanine insertion caused DHFR to become unstructured or inactive. Some “Structure Elements” overlap with “Function Elements”, indicating that loss of structure leads to loss of function. However, other “Structure Elements” are not “Function Elements”, in that alanine insertion mutants of these regions exhibit substrate- or inhibitor-induced folding. There are also some “Function Elements” which are not “Structure Elements”; alanine insertion into these elements deforms the catalytic site topology without the loss of tertiary structure. We hypothesize that these elements are involved essential interactions for structure formation and functional expression. The “Elements” are closely related to the module structure of DHFR. An “Element” belongs to a single module, and a single module is composed of some number of “Elements.” We propose that properties of a module are determined by the “Elements” it contains. Systematic alanine insertion analysis is an effective and unique method for deriving the regions of a sequence that are essential for structure formation and functional expression.

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“…Because of the biological and pharmacological importance of DHFR, dhfr genes from several organisms, including prokaryotes (1,6,9,16,24,25,31,35,36), have been cloned and extensively studied. Some of them have found several applications, e.g., as selection (16) or reporter (17) markers, while the DHFR enzyme from Escherichia coli has been used during leucovorine production (22) or as an ''affinity handle'' (15).…”

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Cloning and molecular analysis of the dihydrofolate reductase gene from Lactococcus lactis

Leszczynska

Bolhuis

Leenhouts

et al. 1995

Appl Environ Microbiol

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The Lactococcus lactis gene encoding trimethoprim resistance has been cloned and expressed in Escherichia coli and Bacillus subtilis. Several lines of evidence indicate that the cloned gene encodes dihydrofolate reductase (DHFR). (i) It fully complements the fol "null" mutation in E. coli. (ii) Nucleotide sequencing of the cloned fragment revealed the presence of one open reading frame encoding a protein that shares homology with the family of bacterial DHFR enzymes. (iii) Overexpression of this open reading frame in E. coli resulted in the appearance in cell extracts of a protein of the expected size as well as in a dramatic increase of DHFR activity. In cell extracts, the DHFR activity was not inhibited by low trimethoprim concentration. By Northern (RNA) blotting and primer extension analyses, the size and the start point of the dhfr transcript, respectively, have been determined. Results of these experiments indicate that in L. lactis the dhfr gene represents part of a larger transcription unit.

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Production of Dihydrofolate Reductase by ClonedEscherichia coliand Its Application to Asymmetric Synthesis ofl-Leucovorin

Cited by 5 publications

References 6 publications

Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid–Base Catalysis

Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid–Base Catalysis

Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase

Cloning and molecular analysis of the dihydrofolate reductase gene from Lactococcus lactis

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