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PDBsum entry 4xby
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References listed in PDB file
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Key reference
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Title
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Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.
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Authors
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Z.Sun,
R.Lonsdale,
X.D.Kong,
J.H.Xu,
J.Zhou,
M.T.Reetz.
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Ref.
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Angew Chem Int Ed Engl, 2015,
54,
12410-12415.
[DOI no: ]
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PubMed id
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Abstract
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Directed evolution based on saturation mutagenesis at sites lining the binding
pocket is a commonly practiced strategy for enhancing or inverting the
stereoselectivity of enzymes for use in organic chemistry or biotechnology.
However, as the number of residues in a randomization site increases to five or
more, the screening effort for 95 % library coverage increases astronomically
until it is no longer feasible. We propose the use of a single amino acid for
saturation mutagenesis at superlarge randomization sites comprising 10 or more
residues. When used to reshape the binding pocket of limonene epoxide hydrolase,
this strategy, which drastically reduces the search space and thus the screening
effort, resulted in R,R- and S,S-selective mutants for the hydrolytic
desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal
structures and docking studies of the mutants unveiled the source of
stereoselectivity and shed light on the mechanistic intricacies of this enzyme.
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