HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 45, 46810-46817, November 5, 2004

This Article Full Text Full Text (PDF) All Versions of this Article: 279/45/46810    most recent M407466200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rui, L. Articles by Wood, T. K. Search for Related Content PubMed PubMed Citation Articles by Rui, L. Articles by Wood, T. K. Social Bookmarking                      What's this?

Active Site Engineering of the Epoxide Hydrolase from Agrobacterium radiobacter AD1 to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene in Cells Expressing an Evolved Toluene ortho-Monooxygenase^*

Lingyun Rui, Li Cao, Wilfred Chen, Kenneth F. Reardon¶, and Thomas K. Wood||

From the Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269–3222, the Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, and the ^¶Department of Chemical Engineering, Colorado State University, Fort Collins, Colorado 80523-1370

Chlorinated ethenes are the most prevalent ground-water pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wild-type EchA at low concentrations (6.8 µM) and up to 10-fold at high concentrations (540 µM). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 µM), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 µM). For complete degradation of cis-DCE to chloride ions, the apparent V_max/K_m for the Escherichia coli strain expressing recombinant the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).

Received for publication, July 6, 2004 , and in revised form, August 18, 2004.

^* This work was supported by National Science Foundation Grants BES-9911469 and BES-0331416. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 860-486-2483; Fax: 860-486-2959; E-mail: twood{at}engr.uconn.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				T. Kadonosono, M. Kato-Murai, and M. Ueda Alteration of substrate specificity of rat neurolysin from matrix metalloproteinase-2/9-type to -3-type specificity by comprehensive mutation Protein Eng. Des. Sel., August 1, 2008; 21(8): 507 - 513. [Abstract] [Full Text] [PDF]

				E. Leonard, K.-H. Lim, P.-N. Saw, and M. A. G. Koffas Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli Appl. Envir. Microbiol., June 15, 2007; 73(12): 3877 - 3886. [Abstract] [Full Text] [PDF]

				A. Herman and D. S. Tawfik Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries Protein Eng. Des. Sel., May 5, 2007; (2007) gzm014v1. [Abstract] [Full Text] [PDF]

				L. Rui, L. Cao, W. Chen, K. F. Reardon, and T. K. Wood Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol Appl. Envir. Microbiol., July 1, 2005; 71(7): 3995 - 4003. [Abstract] [Full Text] [PDF]