De novo design of catalytic proteins

Abstract

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O2-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (kcat͞KM ‫؍‬ 1,500 M ؊1 ⅐min ؊1 ) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.T he de novo design of proteins provides a stringent test of our understanding of their molecular mechanisms of action (1-3). Recently, it has become possible to design proteins with novel three-dimensional structures (4), which has laid the groundwork for the elaboration of function. Catalysis provides a particularly challenging function to achieve, because a successfully designed protein catalyst must bind and precisely orient substrates, transition states, and intermediates adjacent to catalytic groups such as metal ions, general acids, and͞or general bases. Two approaches to the design of catalytic proteins include automated sequence design, in which a novel catalytic site is engineered into a natural protein by mutating a subset of its side chains (5-7), and de novo protein design, which requires the simultaneous design of the entire backbone structure and sequence (2). The first method has the advantage of separating the problem of protein design and folding from the more restricted problem of designing an active site. The second approach has the potential advantage of greater applicability in terms of the sizes and shapes of substrates that can be accommodated. Furthermore, de novo protein design critically tests our understanding of how an amino acid sequence dictates both the folding as well as the activity of a protein.To date, most work on the design of catalytic proteins has focused on hydrolysis of activated 4-nitrophenyl esters, using an active site histidine side chain as a nucleophilic catalyst. Automated sequence design methods have been used to design a variant of thioredoxin that hydrolyses 4-ntirophenyl acetate with a rate enhancement of Ϸ25-fold (7), when the value of k cat ͞K M was compared to the second order rate constant for hydrolysis of 4-methyl imidazole. Proteins with similar or greater catalytic efficiencies were observed frequently in a library of four-helix bundle proteins, whose polar exterior residues were randomly selected from Lys, His, Glu, Gln, Asp, and Asn (8). Baltzer and Nilsson (1) have designed a series of helical bundles that employ His residues to promote hydrolysis and intrapeptide acyl transfer reactions. It has also been possible to design helical bundles that catalyze decarboxylation of oxaloacetate (9), in one case by recognizing a key aldamine intermediate in the reaction (10).Automated sequence design has also been used to design catalytic meta...

Installing hydrolytic activity into a completely de novo protein framework

“…19 By contrast, much less has been demonstrated for the design of completely de novo catalysts; that is, where both the protein scaffold and the catalytic activity are built from scratch. [20][21][22] Although more challenging, this would carry advantages over redesign approaches: firstly, the designer could select and control all, or at least many of the residues, and so engineer the complete construct predictably; secondly, the resulting proteins could be made to function under conditions away from those required by natural proteins; and finally, success in this area would provide the acid test of our understanding of enzyme structure and function. Towards this fully de novo effort, successful designs of hydrolases have included: the decoration of small protein-folding motifs with His residues; 23,24 the employment of Zn 2+ cations as Lewisacidic cofactors; [25][26][27][28] and the identification of catalytically active proteins in combinatorial libraries of sequences patterned to form to all- or all- protein folds.…”

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

“…Although designed proteins that model the structure of native enzymes have been known for a while (4)(5)(6)(7)(8)(9)(10), successful designs of proteins that mimic both the structure and function of native enzymes have been reported only recently (11)(12)(13)(14)(15)(16). While being able to design such functional proteins is laudable, the impact of such an achievement would be greater if the designed proteins can be used to address fundamental issues in chemistry and biology that are difficult to tackle by other methods.…”

Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

“…Various approaches can be used to incorporate functions in the designed protein scaffolds (Smith and Hecht, 2011), and metal binding sites have been a particularly interesting target for this purpose because the biological chemistry of metals is extremely rich (Holm et al, 1996;Lu et al, 2009). For example, by engineering different cofactors like Zn 2+ (Handel et al, 1993), Fe 2+ /Fe 3+ (Kaplan and DeGrado, 2004), heme (Choma et al, 1994) and abiological chromophore (DPP) Zn (Fry et al, 2010) into the de novo designed four-helix bundles, functions like phenol oxidation, electron transfer, and nonlinear optical properties have been obtained.…”