Spiller, Ben and Gershenson, Anne and Arnold, Frances H. and Stevens, Raymond C. (1999) A structural view of evolutionary divergence. Proceedings of the National Academy of Sciences of the United States of America, 96 (22). pp. 12305-12310. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:SPIpnas99
See Usage Policy.
Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:SPIpnas99
Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17°C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 Å, 1.6 Å, and 2.0 Å, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the alpha /beta hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.
|Additional Information:||Copyright © 1999 by The National Academy of Sciences Communicated by William N. Lipscomb, Harvard University, Cambridge, MA, August 30, 1999 (received for review July 2, 1999) We thank Dan Koshland, Judith Klinman, Andrew Mesecar, Melanie Gee, Emily Munsdorff, and D. Borden Lacy for reviewing the manuscript, T. Earnest and L.W. Hung for help at beamline 5.0.2 of the Advance Light Source, P. Kuhn and M. Solstice for help at beamline 7–1 of Stanford Synchrotron Radiation Laboratory. B. Jap and P. Walian for access to in-house x-ray equipment, and D. King for MS. This work was supported in part by the Army Research Office (F.H.A. and A.G.) and by Department of Energy Contract No. DE-AC03-76SF0098 (R.C.S. and B.S.). Data deposition: The coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank, www.rcsb.org [PDB ID code 1QE3 (WT), 1QE8 (organophile 5-c68), and 1C00 (thermophile 8g8)]. 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.|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Archive Administrator|
|Deposited On:||09 Sep 2005|
|Last Modified:||26 Dec 2012 08:40|
Repository Staff Only: item control page