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PDBsum entry 1zdr
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Oxidoreductase
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PDB id
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1zdr
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References listed in PDB file
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Key reference
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Title
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Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues.
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Authors
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H.S.Kim,
S.M.Damo,
S.Y.Lee,
D.Wemmer,
J.P.Klinman.
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Ref.
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Biochemistry, 2005,
44,
11428-11439.
[DOI no: ]
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PubMed id
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Abstract
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Dihydrofolate reductase (DHFR) from a moderate thermophilic organism, Bacillus
stearothermophilus, has been cloned and expressed. Physical characterization of
the protein (BsDHFR) indicates that it is a monomeric protein with a molecular
mass of 18,694.6 Da (0.8), coincident with the mass of 18 694.67 Da calculated
from the primary sequence. Determination of the X-ray structure of BsDHFR
provides the first structure for a monomeric DHFR from a thermophilic organism,
indicating a high degree of conservation of structure in relation to all
chromosomal DHFRs. Structurally based sequence alignment of DHFRs indicates the
following levels of sequence identity and similarity for BsDHFR: 38 and 58% with
Escherichia coli, 35 and 56% with Lactobacillus casei, and 23 and 40% with
Thermotoga maritima, respectively. Steady state kinetic isotope effect studies
indicate an ordered kinetic mechanism at elevated temperatures, with NADPH
binding first to the enzyme. This converts to a more random mechanism at reduced
temperatures, reflected in a greatly reduced K(m) for dihydrofolate at 20
degrees C in relation to that at 60 degrees C. A reduction in either temperature
or pH reduces the degree to which the hydride transfer step is rate-determining
for the second-order reaction of DHF with the enzyme-NADPH binary complex.
Transient state kinetics have been used to study the temperature dependence of
the isotope effect on hydride transfer at pH 9 between 10 and 50 degrees C. The
data support rate-limiting hydride transfer with a moderate enthalpy of
activation (E(a) = 5.5 kcal/mol) and a somewhat greater temperature dependence
for the kinetic isotope effect than predicted from classical behavior [A(H)/A(D)
= 0.57 (0.15)]. Comparison of kinetic parameters for BsDHFR to published data
for DHFR from E. coli and T. maritima shows a decreasing trend in efficiency of
hydride transfer with increasing thermophilicity of the protein. These results
are discussed in the context of the capacity of each enzyme to optimize
H-tunneling from donor (NADPH) to acceptor (DHF) substrates.
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