Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, PA 16802-6300

Contributed by Stephen J. Benkovic

Abstract

The generation of enzymes to catalyze specific reactions is one of the more challenging problems facing protein engineers. Structural similarities between the enzyme scytalone dehydratase with nuclear transport factor 2 (NTF2) suggested the potential for NTF2 to be re-engineered into a scytalone dehydratase-like enzyme. We introduced four key catalytic residues into NTF2 to create a scytalone dehydratase-like active site. A C-terminal helix found in scytalone dehydratase but absent in NTF2 also was added. Mutant NTF2 proteins were tested for catalytic activity by using a spectroscopic assay. One of the engineered enzymes exhibited catalytic activity with minimal k _cat and K _m values of 0.125 min⁻¹ and 800 μM, respectively. This level of catalytic activity represents minimally a 150-fold improvement in activity over the background rate for substrate dehydration and a dramatic step forward from the catalytically inert parent NTF2. This work represents one of the few examples of converting a protein scaffold into an enzyme, outside those arising from the induction of catalytic activity into antibodies.

Footnotes

↵ * Present address: Dyax Corporation, One Kendall Square, Building 600, Cambridge, MA 02139.
↵ † To whom reprint requests should be addressed. e-mail: sjb1{at}psu.edu.
ABBREVIATIONS:

DDBO,

2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one;

NTF2,

nuclear transport factor 2;

quad mutant,

NTF2 containing W41Y, F22Y, F99S, and Q101N mutations;

quad mutant plus flap,

quad mutant plus 20 amino acid C-terminal α-helix from scytalone dehydratase