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PDBsum entry 1fq0
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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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Directed evolution of a new catalytic site in 2-Keto-3-Deoxy-6-Phosphogluconate aldolase from escherichia coli.
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Authors
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N.Wymer,
L.V.Buchanan,
D.Henderson,
N.Mehta,
C.H.Botting,
L.Pocivavsek,
C.A.Fierke,
E.J.Toone,
J.H.Naismith.
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Ref.
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Structure, 2001,
9,
1-9.
[DOI no: ]
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PubMed id
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Abstract
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BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been
identified as useful tools for the organic chemist. However, their utility is
limited in part by their narrow substrate utilization. Site-directed mutagenesis
of various enzymes to alter their specificity has been performed for many years,
typically without the desired effect. More recently directed evolution has been
employed to engineer new activities onto existing scaffoldings. This approach
allows random mutation of the gene and then selects for fitness to purpose those
proteins with the desired activity. To date such approaches have furnished novel
activities through multiple mutations of residues involved in recognition; in no
instance has a key catalytic residue been altered while activity is retained.
RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate
aldolase with reduced but measurable enzyme activity and a synthetically useful
substrate profile. The mutant was identified from directed-evolution
experiments. Modification of substrate specificity is achieved by altering the
position of the active site lysine from one beta strand to a neighboring strand
rather than by modification of the substrate recognition site. The new enzyme is
different to all other existing aldolases with respect to the location of its
active site to secondary structure. The new enzyme still displays enantiofacial
discrimination during aldol addition. We have determined the crystal structure
of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the
double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest
that the scope of directed evolution is substantially larger than previously
envisioned in that it is possible to perturb the active site residues themselves
as well as surrounding loops to alter specificity. The structure of the double
mutant shows how catalytic competency is maintained despite spatial
reorganization of the active site with respect to substrate.
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Figure 1.
Figure 1. The Reaction Catalyzed by KDPG Aldolase
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The above figure is
reprinted
by permission from Cell Press:
Structure
(2001,
9,
1-9)
copyright 2001.
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