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1200 E. California Blvd.
Pasadena, CA
91125-9600
Mail Code: 114-96
Location: 130 Broad
(626) 395-6407
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Research
Fully automated enzyme design can be envisioned as the solution to many complex
synthetic organic chemistry problems. Enzymes are ideal catalysts of organic
reactions because of their extremely high rates of catalysis and their ability
to perform a wide variety of chemical transformations with extreme regio- and
enantioselectivity. With an increasing demand for enantiomerically pure, complex,
biologically active compounds (i.e., drugs), synthetic chemists will have to
look beyond standard chemical strategies in favor of the high yields and strict
selectivity of enzymatic reactions. Designed enzymes will not be restricted to
the reactions accessible by natural enzymes, broadening the range of possible
substrates and products.
Kinetic resolution is a powerful method for transforming a racemic starting material
into an enantiomerically pure product. Our goal is to show that an enzyme can
be designed to carry out a kinetic resolution using the enantioselective hydrolysis
of 2-phenyl-4-benzylphenyloxazolin-5-one (FOX) to produce N-benzoyl-L-phenylalanine
as a model system. As a first step in the design of this enzyme, we will make
a protein capable of selectively binding L-FOX and then design catalytic functionality
into the optimized binding protein.
We have used the ORBIT protein design software to design an optimal binding pocket
around an internally and externally flexible L-FOX structure using the hyperthermophilic T.
thermophilus protein aspartate amino transferase (AspAT) as a scaffold.
Within the context of a poly-alanine scaffold, the location and conformation
of two important binding residues were first chosen based on the geometric constraints
between the ligand and the sidechains that describe “optimal” contacts. The remainder
of the binding pocket residues were repacked around the catalytic residues and
the ligand, resulting in a twelve-fold mutant, named 1GCK-FFBP. We are currently
evaluating the binding of FOX to 1GCK-FFBP using fluorescence anisotropy and
attempting to determine the protein’s effect on the enantiomeric enrichment of
the product. Once binding of the ligand has been established, catalytic residues
that support hydrolysis of the L-enantiomer of FOX can be introduced into the
binding pocket.
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