Chemically Modified “Polar Patch” Mutants of Subtilisin in Peptide Synthesis with Remarkably Broad Substrate Acceptance: Designing Combinatorial Biocatalysts

Abstract

Two structurally unrelated small molecules that stabilize the interaction of a 14–3–3 protein with the proton pump PMA2 have been identified. The compounds are selective among different 14–3–3 protein–protein interactions and are active in vivo. Crystal structures of ternary complexes revealed that the molecules bind to different sites in the interface of the 14–3–3 protein and PMA2 (see picture), thus explaining the different binding kinetics.

Synthesis of copper ion incorporated horseradish peroxidase-based hybrid nanoflowers for enhanced catalytic activity and stability

“…Furthermore, the time-consuming and labor-intensive synthetic procedures have limited application of the chemical modification method. [6][7][8] In contrast to chemical modification, immobilization of enzymes in/on solid supports has offered a simple, alternative, generally requiring one-step reaction. In general, immobilized enzymes show improved stability, making them efficient, reusable and economical.…”

Amination of enzymes to improve biocatalyst performance: coupling genetic modification and physicochemical tools

“…The target 289 enzyme was serine protease subtilisin Bacillus lentus A significant enhancement of the 290 applicability of this enzyme in peptide synthesis was achieved by using the strategy of 291 combined site-directed mutagenesis and chemical modification to create chemically modified 292 mutant enzymes. 96 The introduction of polar and/or homochiral auxiliary substituents, such as 293 X = oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of 294 the primary specificity S 1 pocket created an enzyme with strikingly broad structural substrate 295 specificities. These modified mutante enzymes are capable of catalyzing the coupling 296 reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with 297 glycinamide to give the corresponding dipeptides in good yields.…”

“…303 91% Z-L-Ala-GlyNH 2 and 86% yield of Z-D-Ala-GlyNH 2 using S166C-S-(3R,4S)-304 indenooxazolidinone) was proposed as a tool to allow the use of biocatalysts in parallel library 305 synthesis. 96 …”

Chemical approaches to mapping the function of post‐translational modifications

“…Particularly striking was the broad substrate tolerance that could be engineered (e.g. towards non‐natural amino acids) by appropriate incorporation of the polar domain [47]. In an example that combines the exploration of two modes of modification, ‘polar patch’‐modified enzymes have also been applied to the catalysis of glycan‐modified glycopeptide ligation [36].…”