A Caltech Library Service

Tuning the Activity of an Enzyme for Unusual Environments: Sequential Random Mutagenesis of Subtilisin E for Catalysis in Dimethylformamide

Chen, Keqin and Arnold, Frances H. (1993) Tuning the Activity of an Enzyme for Unusual Environments: Sequential Random Mutagenesis of Subtilisin E for Catalysis in Dimethylformamide. Proceedings of the National Academy of Sciences of the United States of America, 90 (6). pp. 5618-5622. ISSN 0027-8424. PMCID PMC46772.

PDF - Published Version
See Usage Policy.


Use this Persistent URL to link to this item:


Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment-high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60 {rightarrow} Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures-it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1993 by National Academy of Sciences. Communicated by Peter B. Dervan, March 19, 1993 (received for review September 2, 1992) This research was supported by the Office of Naval Research and the Department of Energy (Advanced Industrial Concepts Division). F.H.A. gratefully acknowledges a Presidential Young Investigator award from the National Science Foundation and a fellowship from the David and Lucile Packard Foundation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funding AgencyGrant Number
Office of Naval Research (ONR)UNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
David and Lucile Packard FoundationUNSPECIFIED
Subject Keywords:peptide synthesis, biocatalysis, molecular evolution, serine protease
Issue or Number:6
PubMed Central ID:PMC46772
Record Number:CaltechAUTHORS:CHEpnas93
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:639
Deposited By: Archive Administrator
Deposited On:09 Sep 2005
Last Modified:02 Oct 2019 22:35

Repository Staff Only: item control page