Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts.
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.
Matsumoto
Kazutsugu
K
Department of Chemistry, College of Science & Technology, Meisei University Hodokubo, 2-1-1, Hino, Tokyo 191-8506, Japan.
Davis
Benjamin G
BG
Jones
J Bryan
JB
D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010452:Peptide Biosynthesis; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D019151:Peptide Library; D020860:Subtilisin