2D0K

Methionine-free mutant of Escherichia coli dihydrofolate reductase

2D0K の概要

• エントリーDOI: 10.2210/pdb2d0k/pdb
• 分子名称: Dihydrofolate reductase, CHLORIDE ION, FOLIC ACID, ... (4 entities in total)
• 機能のキーワード: alpha + beta, oxidoreductase, structural genomics
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 36801.54

構造登録者

Katayanagi, K. (登録日: 2005-08-04, 公開日: 2006-02-28, 最終更新日: 2023-10-25)

主引用文献

Iwakura, M.,Maki, K.,Takahashi, H.,Takenawa, T.,Yokota, A.,Katayanagi, K.,Kamiyama, T.,Gekko, K.
Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase
J.Biol.Chem., 281:13234-13246, 2006

PubMed Abstract: 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-angstroms 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.

PubMed: 16510443
DOI: 10.1074/jbc.M508823200

実験手法

X-RAY DIFFRACTION (1.9 Å)