Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts.

Journal article

A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.

Authors: Matsumoto, K; Davis, B; Jones, J

• Publication status: Published
• Journal: Chemistry (Weinheim an der Bergstrasse, Germany)
• Volume: 8
• Issue: 18
• Pages: 4129-4137
• Publication date: 2002-09-01
• DOI: 10.1002/1521-3765(20020916)8:18<4129::aid-chem4129>3.0.co;2-v
• EISSN: 1521-3765
• ISSN: 0947-6539
• Language: English
• Keywords: Combinatorial Chemistry Techniques; Binding Sites; Bacillus; Peptide Library; Subtilisin; Substrate Specificity; Catalysis; Protein Conformation; Mutation; Peptide Biosynthesis; Protein Engineering; Mutagenesis, Site-Directed; Stereoisomerism; Models, Molecular