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PDBsum entry 2d0k
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Oxidoreductase
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PDB id
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2d0k
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References listed in PDB file
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Key reference
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Title
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Evolutional design of a hyperactive cysteine- And methionine-Free mutant of escherichia coli dihydrofolate reductase.
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Authors
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M.Iwakura,
K.Maki,
H.Takahashi,
T.Takenawa,
A.Yokota,
K.Katayanagi,
T.Kamiyama,
K.Gekko.
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Ref.
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J Biol Chem, 2006,
281,
13234-13246.
[DOI no: ]
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PubMed id
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Abstract
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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-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.
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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).
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
13234-13246)
copyright 2006.
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