Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer

Uwajima, Takayuki; Oshiro, Takashi; Eguchi, Tamotsu; Kuge, Yukihiro; Horiguchi, Akira; Igarashi, Ayuko; Mochida, Kenichi; Iwakura, Masanori

doi:10.1016/0006-291x(90)91200-c

Cited by 16 publications

(7 citation statements)

References 5 publications

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“…11 Furthermore, DHFR is a clinically important enzyme not only because it is a target of a number of antifolate drugs, such as methotrexate (MTX) and trimethoprim (TMP), but also because it can be used to produce an anticancer drug, l-leucovorin. 12 The kinetic refolding reaction of DHFR has been characterized by various techniques including stopped-flow circular dichroism (CD) and fluorescence techniques, [13][14][15][16] as well as by pulsed-hydrogen exchange NMR. 17 These previous results showed that within the dead time of the stopped-flow measurement, DHFR accumulates a burst phase intermediate, in which a part of hydrogen bonding network is weakly formed and the complex overall topology of the eight-stranded b-sheet appears to be organized.…”

Section: Introductionmentioning

confidence: 99%

Probing the Interactions between the Folding Elements Early in the Folding of Escherichia coli Dihydrofolate Reductase by Systematic Sequence Perturbation Analysis

Arai

Iwakura

2005

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Probing the Interactions between the Folding Elements Early in the Folding of Escherichia coli Dihydrofolate Reductase by Systematic Sequence Perturbation Analysis

Arai

Iwakura

2005

Journal of Molecular Biology

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“…DHFR is a clinically important enzyme not only as the target of a number of antifolate drugs, such as trimethoprim and methotrexate (8), but also as the enzyme to produce l-leucoverin, an anti-cancer drug, in a stereospecific manner (9). Because overproduction of DHFR in cells makes E. coli trimethoprim-resistant (tmp R ), DHFR has been used not only as a selectable genetic marker, tmp R (10), but also as a handle protein (affinity handle) for the production of peptide (11).…”

mentioning

confidence: 99%

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

Iwakura

Maki

Takahashi

et al. 2006

Journal of Biological Chemistry

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We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine-and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/ M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k cat value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-Å resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.To freely design enzymes with desired properties is the ultimate dream for protein engineers. Because the "protein folding problem," namely how an amino acid sequence determines its tertiary folded structure, has not completely been solved at atomic resolution, the rational design approach is still limited, even for improvement of enzymes from natural sources. Alternatively, protein design can be thought of as a process of "picking up" from all the possible amino acid sequences (i.e. sequence space) until a protein with the desired properties is found, because the structure and function of a protein is determined by its amino acid sequence (1). When we set the goal of protein design "to create a protein of a desired property consisting of a given number of amino acid residues n," the solution can be obtained by the complete search of all the possible amino acid sequences with "n" amino acid residues, the total number reaching 20 n . Therefore, the protein design problem can be reduced to a searching problem in sequence space of polypeptides with n amino acid residues. The solution to this searching problem should include a reliable process that can be performed within a realistic time span, otherwise it is of no use in practical protein design. In this regard, the size of sequence space to be searched is a critical factor (2). The size of the sequence space of a polypeptide with amino acids even as small as 100 is ...

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“…The product (6S)-5 was purified by the method of Temple. 13 The optical purity of compound (6S)-5 was 100% de which was estimated by HPLC (using a Resolvosil column), whereas the optical purity of tetrahydrofolate (6S)-3 prepared in the NADPH recycling system was also 94% de. It might be said that the enhancement of the optical purity could be attributed to the crystallization of compound (6R)-4.…”

Section: Dihydrofolic Acid D Tetrahydrofolic Acidmentioning

confidence: 99%

Large-scale chemoenzymic synthesis of calcium (6S)-5-formyl-5,6,7,8-tetrahydrofolate [(–)-leucovorin] using the NADPH recycling method

Kuge¹,

Inoue²,

Ando³

et al. 1994

J. Chem. Soc., Perkin Trans. 1

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Chemoenzymic large-scale synthesis of the calcium salt of (6s) -5-formyltetrahydrofolic acid [ (-)leucovorin, (6S)-5] was achieved from folic acid 1 via (6s)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DH FR) produced by Zscherichia coli, harbouring a high-expression plasmid, pTP64-1. On the other hand, for the diastereoselective reduction of 7,8-dihydrofolic acid 2 to tetrahydrofolate (6S)-3, a new NADPH recycling system was constructed by coupling with glucose dehydrogenase from Gluconobacter scleroides. Having these enzymic systems to hand, compound 1 was reduced by zinc powder in alkaline solution to give compound 2 which, without isolation, was reduced enzymatically to afford tetrahydrofolate (6s) -3 (94% de). The pH adjustment of the reaction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic reduction. The formed tetrahydrofolate (6s) -

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Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer

Cited by 16 publications

References 5 publications

Probing the Interactions between the Folding Elements Early in the Folding of Escherichia coli Dihydrofolate Reductase by Systematic Sequence Perturbation Analysis

Probing the Interactions between the Folding Elements Early in the Folding of Escherichia coli Dihydrofolate Reductase by Systematic Sequence Perturbation Analysis

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

Large-scale chemoenzymic synthesis of calcium (6S)-5-formyl-5,6,7,8-tetrahydrofolate [(–)-leucovorin] using the NADPH recycling method

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