Searching sequence space for protein catalysts

Genetic selection was used to explore the probability of finding enzymes in protein sequence space. Large degenerate libraries were prepared by replac ing all secondary structure units in a dimeric, helical bundle chorismate m utase with simple binary-patterned modules based on a limited set of four p olar and four nonpolar residues. Two-stage in vivo selection yielded cataly tically active variants possessing biophysical and kinetic properties typic al of the natural enzyme even though approximate to 80% of the protein orig inates from the simplified modules and > 90% of the protein consists of onl y eight different amino acids. This study provides a quantitative assessmen t of the number of sequences compatible with a given fold and implicates pr eviously unidentified residues needed to form a functional active site. Giv en the extremely low incidence of enzymes in completely unbiased libraries, strategies that combine chemical information with genetic selection, like the one used here, may be generally useful in designing novel protein scaff olds with tailored activities.