Abstract
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.
Highlights
Natural evolution produces complex protein folds with a twenty amino acid alphabet
With the exception of a glutamine (Gln88) and three arginines (Arg11, Arg28, Arg51) in the active site and four loop residues (Gly43, Pro45, His66, Val68), all its building blocks belong to the eight-letter alphabet: Asp, Glu, Asn, Lys / Phe, Ile, Leu, Met (Fig. 2A)
Dispensing with eleven of the twenty standard amino acids significantly reduces the diversity of favorable internal packing interactions, leading to destabilization of the overall protein structure
Summary
Natural evolution produces complex protein folds with a twenty amino acid alphabet. Primordial protein synthesis, is believed to have involved only a handful of amino acids [1]. A de novo designed protein constructed from a seven amino acid alphabet adopts a well-defined four-helix bundle fold [5]. Such simplified proteins are generally devoid of function. 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]. We extend this work and show that fully functional proteins can be constructed entirely from a nine-amino acid set. These represent the most severely simplified enzymes reported to date
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