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PDBsum entry 1rxh
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Unknown function
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PDB id
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1rxh
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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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Structural elements responsible for conversion of streptavidin to a pseudoenzyme.
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Authors
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Y.Eisenberg-Domovich,
Y.Pazy,
O.Nir,
B.Raboy,
E.A.Bayer,
M.Wilchek,
O.Livnah.
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Ref.
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Proc Natl Acad Sci U S A, 2004,
101,
5916-5921.
[DOI no: ]
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PubMed id
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Abstract
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Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild
alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH
12. Recently, we imposed hydrolytic activity on streptavidin by rational
mutagenesis, based on the molecular elements responsible for the hydrolysis by
avidin. Three mutants were designed, whereby the desired features, the
distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the
combined mutation (M3), were transferred from avidin to streptavidin. The
crystal structures of the mutants, in complex with biotinyl p-nitroanilide
(BNA), the stable amide analogue of BNP, were determined. The results
demonstrate that the point mutation alone has little effect on hydrolysis, and
BNA exhibits a conformation similar to that of streptavidin. Substitution of a
lengthier L3,4 loop (from avidin to streptavidin), resulted in an open
conformation, thus exposing the ligand to solvent. Moreover, the amide bond of
BNA was flipped relative to that of the streptavidin and M1 complexes, thus
deflecting the nitro group toward Lys-121. Consequently, the leaving group
potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at
pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was
required. The combination of loop replacement and point mutation served to
further increase the leaving group potential by interaction of the nitro group
with Arg-124 and Lys-121. The information derived from this study may provide
insight into the design of enzymes and transfer of desired properties among
homologous proteins.
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Figure 1.
Fig. 1. Electron-density maps of M1, M2, and M3 in complex
with BNA. Shown are stereoviews of F[o] - F[c] electron-density
maps calculated at respective resolutions indicated in Table 1,
after the initial cycle of refinement with no ligand in the
model. The initial model included leucine at position 124, and
for the M1 and M3 complexes the extended electron density
clearly suggests the existence of an arginine residue at this
position. The maps were constructed at 2.0  with superimposed
coordinates from the final models.
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Figure 3.
Fig. 3. Tube representation of the tertiary structures of
M3 (blue) and streptavidin (magenta) complexes with BNA (shown
in black). The L3,4 loop in both molecules is shown in gold. In
streptavidin, the loop is in the closed conformation, thus
burying the BNA ligand almost completely. In M3, where the loop
has three more residues, L3,4 adopts an open conformation. Note
the flipping of the amide bond of the ligand and the altered
conformation of the p-nitroanilide group.
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