Designing the substrate specificity of d-hydantoinase using a rational approach

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters

“…While a rational amino acid exchange at a position identified by molecular docking was successful in the past (Höst and Jonsson, 2008;Lee et al, 2009), the amino acid exchanges from library I, which was constructed in a similar way, did not successfully increase the activity for bulky acyl substrates in the case of CALB. With a bulky and hydrophobic substrate we modeled enzyme variant with a single amino acid exchange that featured smaller and uncharged amino acids in the hotspot positions and expressed selected enzyme variants that displayed small improvements in the docking score in E. coli.…”

“…The identification of amino acid positions that are relevant for the conversion of a specific substrate has been successfully shown for a carbonyl reductase (Zhu et al, 2008), a fructosyl amine oxidase (Miura et al, 2008), and a glutathione transferase (Kapoli et al, 2008). In contrast to the simple prediction of hotspots by investigating the enzyme structure, a specific amino acid exchanges with increased catalytic activity for specific substrates were predicted for the human anhydrase II (Höst and Jonsson, 2008) and a hydantoinase (Lee et al, 2009) by molecular docking. In this study, molecular docking was first used to identify hotspots for mutagenesis in CALB wild type, then two strategies were followed: (i) Single amino acid exchange variants were modeled, collected in a library I, and selected by their docking scores and (ii) a range of amino acids exchanges for these hotspots were chosen in order to create more space for the substrates in the binding pocket and to establish a biochemical environment favorable for the substrates, a combinatorial in silico library II of 2400 CALB variants was built, and substrate-imprinted docking (Juhl et al, 2009) was applied to identify amino acid exchanges that frequently resulted in better docking scores.…”

From directed evolution to computational enzyme engineering—A review

“…The best design exhibited a 27‐fold improvement in NADH‐dependent activity. Other successes from the same group include, OptGraft for grafting a binding site from one protein into another protein scaffold, rational design to obtain 200‐fold higher D‐hydantoinase activity in Bacillus stearothermophilus using just two amino acid changes, OptZyme for redesigning enzymes by improving binding to a transition state analogue instead of the substrate as it correlates with greater turnover, IPRO Suite of programs for fully‐automated protein redesign, and altering substrate specificity of thioesterase enzyme from long‐chain fatty acyl ACP to medium‐chain ones …”

Computational screening of novel thiamine-catalyzed decarboxylation reactions of 2-keto acids

“…Computational tools are increasingly used to complement experimental approaches for metabolic engineering [3][4][5][6][7][8][9][10][11][12]. One approach that has emerged recently utilizes a computational framework based on graph theory that creates complex networks of reactions and compounds based on generalized enzyme reactions [13][14][15][16][17][18][19][20].…”