An Active Enzyme Constructed from a 9-Amino Acid Alphabet

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.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...

Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

“…4 Several researchers have demonstrated that the amino acid usage of various natural globular proteins and enzymes can be restricted to 5-13 members without substantial alteration of their structures and biological functions. [5][6][7][8] Riddle et al simplified the sequence of a small b-sheet protein, the SH3 domain, by using phage display selection, and produced two SH3 variants in which 90% of the sequence, excluding the binding region, utilized only five amino acids (Ala, Gly, Glu, Ile, and Lys). 5 Silverman et al generated variants of the prototypical (b/a) 8 barrel enzyme, triosephosphate isomerase (TIM), in which the amino acids at 142 of 182 structural positions were simplified to seven kinds (Ala, Glu, Val, Lys, Phe, Leu, and Gln) by means of in vivo selection for TIM activity.…”

Simplification of complexity in protein molecular systems by grouping amino acids: a view from physics

“…Therefore, to design the proteins based on a simplified alphabet would provide a way to check the validity of the simplified alphabet. This kind of idea has been realized in many experiments [56][57][58][59][60][61][62][63]. Facing with the helix bundles, de novo designs were realized based on hydrophobic/polar patterns along the protein chains [56,57].…”

“…To reproduce the catalytic activity, more types of amino acids are needed. Through sophisticated designs and selections, active enzymes could be constructed from 9-amino-acid alphabets [61][62][63]. It reflects that there are more demands with further function requirements.…”

Evolutionary Engineering of Artificial Proteins with Limited Sets of Primitive Amino Acids

“…Moreover, artificial proteins that form well-folded structures after interaction with their target were produced (Walter et al, 2005;Vamvaca et al, 2004;Chaput & Szostak, 2004). Such partially structured polypeptides might have been the first evolutionary intermediates, and their functions and structures would have coevolved (Tokuriki & Tawfik, 2009b).…”

“…Akanuma et al (2002) generated variants of orotate phosphoribosyl transferase in which 88% of the sequence used just nine types of amino acids. Finally, Walter et al (2005) created an active enzyme, chorismate mutase, which was constructed entirely from nine types of amino acids. Other researchers have attempted to produce de novo proteins from designed combinatorial libraries.…”