RETRACTED: Local Encoding of Computationally Designed Enzyme Activity

Abstract

One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously we have shown that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10 5 -10 6 -fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is locally encoded within the layer of residues directly in contact with the substrate, and that in this case at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

A novel method for enzyme design

“…12 In the present method, candidate residue combinations matching the natural residues are searched for in the cavity of a scaffold protein. The matching includes Ca-[ Cb vectors as well as key side-chain atoms.…”

Molecular Design of Specific Metal‐Binding Peptide Sequences from Protein Fragments: Theory and Experiment

“…Keywords: ab initio calculations · mass spectrometry · metal-ion chelation · molecular design · peptides include the removal of metals from polluted environments, either by bacterial strains [1][2][3][4][5] or other biotechnological techniques, [6][7][8] the design of novel biosensors, [9][10][11] the redesign of proteins (to provide new building blocks or maquettes) [12,13] or the design of new chelating compounds for medicinal chemistry. [14] Numerous successful attempts have recently been made to alter the metal-binding properties of selected proteins with the aims of modifying protein function, [15][16][17][18][19][20] engineering novel metalloproteins or metal-binding sites in natural metalloproteins [21][22][23][24][25][26] or tuning metalloprotein or peptide specificity towards a particular metal ion. [27][28][29][30][31] While most studies employ the standard repertoire of amino acids, one approach has been to include unnatural amino acids or nonnative metal-containing cofactors.…”