High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

Changchien, Li-Ming; Garibian, Araik; Frasca, Verna; Lobo, Angelo P.; Maley, Gladys F.; Maley, Frank

doi:10.1006/prep.2000.1245

Cited by 31 publications

(36 citation statements)

References 21 publications

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“…Ultima Gold liquid scintillation cocktails were from Packard Bioscience. Liquid scintillation vials were from Research Products International Corp. [2- 3 H]iPrOH and [2- 2 H]iPrOH were prepared by reduction of acetone with [ 3 H]NaBH 4 and [ 2 H]NaBH 4 , respectively.68 Dihydrofolate (H 2 folate) was synthesized using Blakely’s method.69 The WT 70 and Y209W 41 ecTSase enzymes were expressed and purified following the previously published procedures. All other materials were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

et al. 2012

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The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of E. coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between those two crystal structures is in the anisotropic B-factors, which indicates disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature-dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and acceptor at the tunneling ready state. These observations suggest that that the mutation disrupts the concerted motions that bring the H-donor and acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different timescales on the hydride transfer reaction within a complex enzymatic mechanism.

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Section: Methodsmentioning

confidence: 99%

A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

et al. 2012

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“…The expression system for E. coli TS was a generous gift from R. Stroud, University of California at San Francisco. The enzyme was purified following the procedure of Changchien et al 18 All other materials were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

“…Labeling substrates or cofactors at primary (18) hydrogen (hydrogen that is involved in C-H bond cleavage) or secondary (28) hydrogen (another hydrogen on the same carbon) with multiple hydrogen isotopes is of general interest. Such labeling has been used to measure competitive KIEs in many biological systems.…”

Section: Introductionmentioning

confidence: 99%

Microscale synthesis of isotopically labeled 6R‐N⁵, N¹⁰ methylene‐5, 6, 7, 8‐tetrahydrofolate

Hong

Kohen

2005

Labelled Comp Radiopharmac

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“…Wild-type E. coli TS was prepared and purified according to the procedure of ref (34). Y94F was prepared and purified as described in detail elsewhere (6,13,19).…”

Section: Enzymementioning

confidence: 99%

Role of Y94 in Proton and Hydride Transfers Catalyzed by Thymidylate Synthase

2007

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Thymidylate synthase (TS) catalyzes the substitution of a carbon-bound proton in a uracil base by a methyl group to yield thymine in the de novo biosynthesis of this DNA base. The enzymatic mechanism involves making and breaking several covalent bonds. Traditionally, a conserved tyrosine (Y94 in E. coli, Y146 in L. casei, and Y135 in human) was assumed to serve as the general base catalyzing the proton abstraction. That assumption was examined here by comparing the nature of the proton abstraction using wild type (wt) E. coli TS (ecTS) and its Y94F mutant (with a two orders of magnitude reduced turnover rate). A subsequent hydride transfer was also studied using the wt and Y94F. The physical nature of both H-transfer steps was examined by determining intrinsic kinetic isotope effects (KIEs). Surprisingly, the findings did not suggest a direct role for Y94 in the proton abstraction step. The effect of this mutation on the subsequent hydride transfer was examined by a comparison of the temperature dependency of the intrinsic KIE on both the wt and the mutant. The intrinsic KIEs for Y94F at physiological temperatures were slightly smaller than for wt, but otherwise, were as temperature independent, suggesting a perfectly pre-organized reaction coordinate for both enzymes. At reduced temperature, however, the KIE for the mutant increased with decreasing temperature, indicating a poorly pre-organized reaction coordinate. Other kinetic and structural properties were also compared and the findings suggested that Y94 is part of a H-bond network that plays a critical role at a step between the proton and the hydride transfers, presumably the dissociation of H 4 folate from the covalently bound intermediate. The possibility that no single residue serves as the general base in question, but rather, that the whole network of H-bonds at the active site catalyzes proton abstraction, is discussed.Thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridylate (dUMP) with 5,10-methylene−5, 6, 7, 8-tetrahydrofolate (CH 2 H 4 folate), forming thymidine monophosphate (dTMP) and 7, 8-dihydrofolate (H 2 folate) (1). TS activity is essential to living organisms since it catalyzes the de novo synthesis of one of the DNA building blocks. Consequently, TS is a common target in cancer chemotherapy, antibiotic drugs, and gene therapy (2,3). The TS-catalyzed reaction has been elucidated in detail by a wide variety of kinetic, genetic, and structural methodologies (1,(4)(5)(6), which have shown that TS is a homodimer utilizing a half-of-the-sites-activity mechanism (7,8). Steady state measurements indicated a bi-bi ordered mechanism with substrate (dUMP) binding before the CH 2 H 4 folate (4,9). Kinetic and structural studies identified coherent protein motion that appear coupled to a hydride transfer step (10,11), which is rate limiting for the wt TS. † This work was supported by NIH R01 GM65368-01 and NSF CHE-0133117 to A.K. *Address correspondence to this author: Tel: 319-335-0234. Fax: 319-335-1270, Email: amn...

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High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

Cited by 31 publications

References 21 publications

A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

Microscale synthesis of isotopically labeled 6R‐N⁵, N¹⁰ methylene‐5, 6, 7, 8‐tetrahydrofolate

Role of Y94 in Proton and Hydride Transfers Catalyzed by Thymidylate Synthase

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High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

Cited by 31 publications

References 21 publications

A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

A Remote Mutation Affects the Hydride Transfer by Disrupting Concerted Protein Motions in Thymidylate Synthase

Microscale synthesis of isotopically labeled 6R‐N5, N10 methylene‐5, 6, 7, 8‐tetrahydrofolate

Role of Y94 in Proton and Hydride Transfers Catalyzed by Thymidylate Synthase

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Microscale synthesis of isotopically labeled 6R‐N⁵, N¹⁰ methylene‐5, 6, 7, 8‐tetrahydrofolate