Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.Natural evolution produces complex protein folds with a 20-amino acid alphabet. Primordial protein synthesis, however, is believed to have involved only a handful of amino acids (1). What is the minimal number of building blocks required for protein structure and function? Several studies have demonstrated that considerably reduced amino acid alphabets are sufficient to encode native-like proteins (2-5). For instance, a de novo designed protein constructed from a 7-amino acid alphabet adopts a well defined four-helix bundle fold (5). Nevertheless, such simplified proteins are generally devoid of function.Simplifying existing proteins without impairing their normal function is challenging, too, given the precise identity and positioning of residues required for binding and catalysis. Selection strategies in combination with design have been successfully applied to search sequence space for active proteins (6 -8). For example, phage display has been exploited to obtain functional SH3 domain variants in which 70% of the wild-type sequence was replaced with a five-letter amino acid alphabet while retaining key binding site residues (6).In a previous study, we exploited selection in vivo to replace all secondary structure units in an AroQ chorismate mutase (CM) 2 with simple binary-patterned modules of 4 polar and 4 apolar residues (9). However, the total number of amino acid types present in the resulting CMs was 14 since the highly conserved active site amino acids and the loop residues were held constant in the original design. Here, we have extended this work and shown that fully functional proteins can be constructed entirely from a 9-amino acid set. These represent the most severely simplified enzymes reported to date. MATERIALS AND METHODSReagents-Restriction enzymes were from New England Biolabs. T4 DNA ligase was from Fermentas. Pfu Turbo polymerase was from Stratagene. Protein concentration was determined with the Coomassie Plus protein assay reagent (Pierce), using bovine serum albumin as the calibration standard.Library Construction-Libraries were constructed by combinatorial site-directed mutagenesis. N-and C-terminal fragments were amplified from plasmid pKT-3 containing the parent CM gene (9) using outside primers sspREX03 (21 bp, 5Ј-CATCCGGC...

Genetic selection was used to explore the probability of finding enzymes in protein sequence space. Large degenerate libraries were prepared by replacing all secondary structure units in a dimeric, helical bundle chorismate mutase with simple binarypatterned modules based on a limited set of four polar and four nonpolar residues. Two-stage in vivo selection yielded catalytically active variants possessing biophysical and kinetic properties typical of the natural enzyme even though Ϸ80% of the protein originates from the simplified modules and >90% of the protein consists of only eight different amino acids. This study provides a quantitative assessment of the number of sequences compatible with a given fold and implicates previously unidentified residues needed to form a functional active site. Given 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 scaffolds with tailored activities. Despite recent progress on the de novo design of structurally defined proteins (1-4), creation of stable scaffolds with tailored enzymatic activities remains an unrealized challenge. Not only is our understanding of the relationship between sequence, structure, and function incomplete, but the requirement for catalysis imposes severe constraints on design. Misplacement of catalytic residues by even a few tenths of an angstrom can mean the difference between full activity and none at all.Direct selection of catalysts from pools of fully randomized polypeptides is a conceivable alternative to de novo design, requiring no foreknowledge of structure or mechanism. An analogous approach has yielded RNA catalysts for a variety of chemical reactions (5). However, a 100-residue protein has 20 100 (1.3 ϫ 10 130 ) possible sequences. Even a library with the mass of the Earth itself-5.98 ϫ 10 27 g-would comprise at most 3.3 ϫ 10 47 different sequences, or a miniscule fraction of such diversity. Unless protein catalysts are unexpectedly abundant and evenly distributed in sequence space, such a strategy will clearly be impractical.Combination of these two approaches represents a potentially attainable middle ground. For example, basic structural information, such as the sequence preferences of helices and sheets or the tendency of hydrophobic residues to be buried in the protein interior, might be used to design focused libraries from which catalysts could be selected with reasonable probability. In fact, binary patterning of polar and nonpolar amino acids (6-8) has been used to generate four-helix bundle proteins (9) that exhibit some native-like properties, including protease resistance and cooperative unfolding (10, 11).Here we show that a combinatorial approach that couples modular design and selection can successfully reproduce a known catalytic activity with an unnatural sequence based on a severely restricted set of building blocks. Specifically, we have constructed large bi...

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.Keywords: chorismate mutase; directed evolution; genetic selection; DNA shuffling; quaternary structure A large fraction of proteins in living cells exist as symmetric assemblies of multiple subunits (Goodsell and Olson 2000). Oligomerization provides advantageous properties often not available to the monomer, such as increased stability, new intersubunit binding sites, cooperativity, and allosteric regulation. To understand the chemical and genetic basis for the evolutionary transition between monomers and oligomers, several studies have focused on altering the quaternary structure of existing proteins by either reducing or increasing the number of their subunits (Mossing and Sauer 1990;Green et al. 1995;Dickason and Huston 1996; MacBeath et al. 1998b,c).In nature, infrequent but beneficial mutations are identified and propagated by genetic selection. This tool can also be exploited effectively in the laboratory to obtain novel protein oligomers starting from a diverse population of variants (Taylor et al. 2001a;Woycechowsky and Hilvert 2004). It is especially valuable in the case of enzymes, where structural changes are often poorly tolerated, and mutations that alter quaternary structure without causing a significant loss in catalytic activity are relatively rare.We have successfully used genetic selection to alter the topology of homodimeric AroQ chorismate mutases (CMs)-domain-swapped enzymes that catalyze a key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. For example, by inserting a random hinge-loop of eight residues into the H1 helix of the thermostable mutase from Methanococcus jannaschii (MjCM) and selecting for functional variants by genetic complementation, we obtained a monomeric catalyst with near native activity (MacBeath et al. 1998c). The topologically redesigned monomer unexpectedly exhibits the properties of a molten globule (Vamvaca et al. 2004), but structural ordering seen upon ligand binding indicates that protein folding and catalysis can be efficiently coupled.Reprint requests to: Donald Hilvert, Laboratorium fu¨r Organische Chemie, ETH Ho¨nggerberg/HCI F 339, CH-8093 Zu¨rich/Switzerland; e-mail: hilvert@org.chem.ethz...

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