PDBsum entry 2d0k

Oxidoreductase

PDB id: 2d0k

Contents

Protein chains

159 a.a. *

Ligands

FOL ×2

Metals

_CL ×2

Waters ×206

* Residue conservation analysis

PDB id:

2d0k

Name:

Oxidoreductase

Title:

Methionine-free mutant of escherichia coli dihydrofolate reductase

Structure:

Dihydrofolate reductase. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.90Å R-factor: 0.205 R-free: 0.284

Authors:

K.Katayanagi

Key ref:

M.Iwakura et al. (2006). Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase. J Biol Chem, 281, 13234-13246. PubMed id: 16510443 DOI: 10.1074/jbc.M508823200

Date:

04-Aug-05 Release date: 28-Feb-06

Protein chains

P0ABQ4  (DYR_ECOLI) -  Dihydrofolate reductase from Escherichia coli (strain K12)

Seq: 159 a.a.

Struc: 159 a.a.*

* PDB and UniProt seqs differ at 7 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.5.1.3 - dihydrofolate reductase.
• Pathway: Folate Coenzymes
• Reaction: (6S)-5,6,7,8-tetrahydrofolate + NADP⁺ = 7,8-dihydrofolate + NADPH + H⁺

(6S)-5,6,7,8-tetrahydrofolate + NADP(+) = 7,8-dihydrofolate (Bound ligand (Het Group name = FOL) corresponds exactly) + NADPH + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M508823200

J Biol Chem 281:13234-13246 (2006)

PubMed id: 16510443

Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase.

M.Iwakura, K.Maki, H.Takahashi, T.Takenawa, A.Yokota, K.Katayanagi, T.Kamiyama, K.Gekko.

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.

Selected figure(s)

Figure 4.
FIGURE 4. Displacement of the crystal structure between the wild type (Protein Data Bank code 1DYI; cyan) and ANLYF (magenta). The r.m.s.d. calculated by using the backbone atoms of the adenosine binding subdomain, the loop subdomain, and folate of the two crystal structures was minimized in A-C, respectively. The regions used for calculating r.m.s.d. are colored explicitly in gray. D and E show representative residues located in the vicinity of the axis of the hinge motion, in which Met-42 (Tyr-42) (green) and Met-92 (Phe-92) (orange) are also included for the wild type and ANLYF, respectively. Folates colored blue and red are bound with the wild type and ANLYF, respectively. All the panels were drawn using a program MOLMOL (66).

Figure 10.
FIGURE 10. Plot of logarithms of the relative activity (k[cat] of mutant protein/k[cat] of the wild type) of ANLYF (•) and the mutant proteins created at sites 67, 121, and 145 ( ) of DHFR against their adiabatic compressibilities, 1 . Data for the mutants except ANLYF were taken from Ref. 34.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 13234-13246) copyright 2006.

Figures were selected by an automated process.