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PDBsum entry 1l9r
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Oxidoreductase
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PDB id
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1l9r
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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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Directing the mode of nitrite binding to a copper-Containing nitrite reductase from alcaligenes faecalis s-6: characterization of an active site isoleucine.
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Authors
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M.J.Boulanger,
M.E.Murphy.
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Ref.
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Protein Sci, 2003,
12,
248-256.
[DOI no: ]
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PubMed id
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Abstract
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Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism
of copper-containing nitrite reductases remains controversial. A key source of
controversy is the productive binding mode of nitrite in the active site. To
identify and characterize the molecular determinants associated with nitrite
binding, we applied a combinatorial mutagenesis approach to generate a small
library of six variants at position 257 in nitrite reductase from Alcaligenes
faecalis S-6. The activities of these six variants span nearly two orders of
magnitude with one variant, I257V, the only observed natural substitution for
Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8
A) nitrite-soaked crystal structures of these variants display different modes
of nitrite binding that correlate well with the altered activities. These
studies identify for the first time that the highly conserved Ile257 in the
native enzyme is a key molecular determinant in directing a catalytically
competent mode of nitrite binding in the active site. The O-coordinate bidentate
binding mode of nitrite observed in native and mutant forms with high activity
supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic
coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)],
AfNiR have been deposited
in the Protein Data Bank [PDB identification codes are listed in Table 2].)
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Figure 1.
Figure 1. Crystal structures of the six nitrite-soaked I257
variants in the upper six panels. Hydrogen bonds are shown as
dashed gray lines with ligand bonds drawn in solid, dark gray
lines. Water molecules are drawn as aquamarine spheres. Copper
atoms are gray; nitrogen atoms are dark blue, oxygen atoms are
red, and sulfur atoms are yellow. The backbone of monomers B and
C are shown in burgundy and in teal, respectively. Bonds of the
nitrite molecule bound in the active site are white. Omit Fo-Fc
electron density maps are contoured at 4  and drawn as
a green wire mesh. Panels A and B depict the multiple
conformations of the nitrite bound in the active site of the
Ile257 variants and including nitrite bound to the oxidized D98N
(black atoms) and H255N (gray atoms) crystal structures
(Boulanger and Murphy 2001). With the exception of the purple
nitrite molecules in panel B, nitrite molecules are blue to red
with increasing specific activity: Red, I257V[NO[2]^-]; orange,
native NiR from Alcaligenes faecalis S-6; light orange,
I257L[NO[2]^-]; yellow, I257M[NO[2]^-]; green, I257A[NO[2]^-];
cyan, I257G[NO[2]^-]; blue, I257T[NO[2]^-].
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The above figure is
reprinted
by permission from the Protein Society:
Protein Sci
(2003,
12,
248-256)
copyright 2003.
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