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PDBsum entry 2e0x
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References listed in PDB file
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Key reference
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Title
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Crystal structure of the gamma-Glutamyltranspeptidase precursor protein from escherichia coli. Structural changes upon autocatalytic processing and implications for the maturation mechanism.
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Authors
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T.Okada,
H.Suzuki,
K.Wada,
H.Kumagai,
K.Fukuyama.
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Ref.
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J Biol Chem, 2007,
282,
2433-2439.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
perfect match.
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Abstract
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Gamma-glutamyltranspeptidase (GGT) is an extracellular enzyme that plays a key
role in glutathione metabolism. The mature GGT is a heterodimer consisting of L-
and S-subunits that is generated by posttranslational cleavage of the peptide
bond between Gln-390 and Thr-391 in the precursor protein. Thr-391, which
becomes the N-terminal residue of the S-subunit, acts as the active residue in
the catalytic reaction. The crystal structure of a mutant GGT, T391A, that is
unable to undergo autocatalytic processing, has been determined at 2.55-A
resolution. Structural comparison of the precursor protein and mature GGT
demonstrates that the structures of the core regions in the two proteins are
unchanged, but marked differences are found near the active site. In particular,
in the precursor, the segment corresponding to the C-terminal region of the
L-subunit occupies the site where the loop (residues 438-449) forms the lid of
the gamma-glutamyl group-binding pocket in the mature GGT. This result
demonstrates that, upon cleavage of the N-terminal peptide bond of Thr-391, the
newly produced C terminus (residues 375-390) flips out, allowing the 438-449
segment to form the gamma-glutamyl group-binding pocket. The electron density
map for the T391A protein also identified a water molecule near the carbonyl
carbon atom of Gln-390. The spatial arrangement around the water and Thr-391
relative to the scissile peptide bond appears suitable for the initiation of
autocatalytic processing, as in other members of the N-terminal nucleophile
hydrolase superfamily.
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Figure 1.
FIGURE 1. A stereo view of the F[o] – F[c] omit map
around the processing site (A molecule). The map was generated
on the basis of F[c] calculated from the model, which was
derived from the refinement using REFMAC5 (23) omitting residues
385–392 and the water molecule (W4). The map was contoured at
the 2.5  level. A ball-and-stick
model of the T391A protein is overlaid on the map. The arrow
indicates the scissile peptide bond that is cleaved in the
wild-type precursor protein (Gln-390 to Thr-391). The figure was
prepared using PYMOL (31).
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Figure 2.
FIGURE 2. The tertiary structure of the T391A protein. A, a
ribbon drawing of the T391A protein (B molecule). The segments
in the T391A protein that correspond to the L- and S-subunits
are pink and green, respectively, and the P-segment (residue
375–390) is highlighted in orange. Terminal residues that
generate invisible segments are labeled. The orange arrow
indicates the site at which autocatalytic processing occurs. B,
a stereo view of the superimposition of C  traces of the T391A
protein and mature GGT. The structure of mature GGT (A molecule
of SeMet-GGT in (19)) was superimposed on that of the T391A
protein (B molecule). P-segment residues in the T391A protein
and in mature GGT are orange and blue, respectively. Residues
that had C atoms displaced by >1
Å upon processing are in yellow. Residues of mature GGT
that are invisible in the T391A protein are shown in black.
Regions of invisible residues are circled in green. The distance
between Ser-387 C and Thr-391 N in mature GGT is shown. B is
rotated by 30° around the vertical axis relative to A. C, a
close-up view of the segment Glu-377 to Pro-380. A stick model
of mature GGT (blue) is superimposed on the T391A protein
(orange). The figures were prepared using PYMOL (31).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
2433-2439)
copyright 2007.
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Secondary reference #1
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Title
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Crystal structures of gamma-Glutamyltranspeptidase from escherichia coli, A key enzyme in glutathione metabolism, And its reaction intermediate.
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Authors
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T.Okada,
H.Suzuki,
K.Wada,
H.Kumagai,
K.Fukuyama.
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Ref.
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Proc Natl Acad Sci U S A, 2006,
103,
6471-6476.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Structure of E. coli GGT. (a) Ribbon drawing of the
GGT heterodimer. The L subunit is colored blue, and the S
subunit is colored green. (b) Ribbon drawing of the L subunit.
(c) Ribbon drawing of the S subunit.  -Helices and  -strands
are labeled. In each of the L and S subunits, the N terminus is
blue and the C terminus is red, with intermediate colors
following the distance in the sequence from the N terminus. The
N-terminal residue of the S subunit (Thr-391) is shown with a
stick model. (d) A topology diagram of E. coli GGT. Circle,
triangle, and square indicate -helix, -strand,
and insertion not conserved among Ntn-hydrolases, respectively.
The secondary structures were defined with DSSP (19). The
figures were prepared with PYMOL (20) and TOPS (21).
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Figure 2.
Fig. 2. The structure of the substrate binding pocket of E.
coli GGT. (a) Surface drawing of substrate binding pocket. The
stick model of the  -glutamyl moiety,
nucleophile (Thr-391), and residues forming the wall (Asn-411
and Tyr-444) are shown in blue, green, and yellow, respectively.
Green dots represent the groove in which the peptide of the
precursor protein is assumed to be present. The hydrogen bond
between Asn-411 O  and Tyr-444 O  is shown
as a dashed line. The ribbon model shown in yellow represents
residues Pro-438–Gly-449, which are absent in B. subtilis GGT.
(b) The (F[o] – F[c]) omit map contoured at the 3  level
for GGT- G. The omit map was
generated by omitting the -glutamyl moiety,
Thr-391, and a water molecule (labeled W2) from the model.
Ball-and-stick models of -glutamyl–enzyme
complex are overlaid on the map. The residues involved in
substrate binding and enzyme reaction are shown in the model.
For the clarity, the side chains of Gln-89, Leu-410, and Thr-412
are omitted from the model. Water molecules involved in
substrate binding and the catalytic reaction are labeled (W1
 W3).
The hydrogen bonds are shown as dashed lines. (c) The (F[o] –
F[c]) omit map for GGT-Glu prepared as for GGT- G. The
view direction is rotated by 40° around the vertical axis
relative to b. The figures were prepared with PYMOL (20).
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