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PDBsum entry 1ocv
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Ketosteroid isomerase
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PDB id
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1ocv
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Origin of the different ph activity profile in two homologous ketosteroid isomerases.
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Authors
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Y.S.Yun,
T.H.Lee,
G.H.Nam,
D.O. .S.Jang,
S.Shin,
B.H.Oh,
K.Y.Choi.
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Ref.
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J Biol Chem, 2003,
278,
28229-28236.
[DOI no: ]
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PubMed id
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Abstract
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Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni
(TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity
profiles. TI-WT loses activity below pH 5.0 due to the protonation of the
conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural
analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a
hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced
with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W
mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared
kinetic parameters of those mutants at different pH levels. PI-W116F exhibited
significantly decreased catalytic activity at acidic pH like TI-WT, whereas
TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the
kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal
structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could
form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The
present results demonstrate that the activities of both PI-WT and TI-F116W at
low pH were maintained by a tryptophan, which was able not only to lower the pKa
value of the catalytic base but also to increase the substrate affinity. This is
one example of the strategy nature can adopt to evolve the diversity of the
catalytic function in the enzymes. Our results provide insight into deciphering
the molecular evolution of the enzyme and creating novel enzymes by protein
engineering.
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Figure 2.
FIG. 2. pH dependence of kinetic parameters of WT and
mutant KSIs. The lines represent nonlinear least-squares fits of
the data to Equations 1 and 2 to obtain the pK[E] and pK[ES]
values, respectively, as listed in Table I. 5,10-EST was used as
a substrate for graphs C and D, and for PI-WT in graphs A and B.
5-AND was used as a substrate for graphs E and F, and for
PI-W116F in graphs A and B.
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Figure 4.
FIG. 4. Stereoview of the catalytic base of TI-F116W with
2F[o]-F[c]-simulated annealing omit electron density map
contoured at 1.0  . Residues Trp-116 and
Asp-38, which were omitted from the model, display clear
electron density. A hydrogen bond between Asp-38 and Trp-116 is
represented by a dashed line. The figure was drawn by using the
program BobScript and rendered using Raster3D.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2003,
278,
28229-28236)
copyright 2003.
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