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Journal of Bacteriology, March 2000, p. 1641-1649, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

Rebecca E. Parales,* Kyoung Lee,dagger Sol M. Resnick,Dagger Haiyan Jiang,§ Daniel J. Lessner, and David T. Gibson

Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242

Received 22 September 1999/Accepted 14 December 1999

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

* Corresponding author. Mailing address: Department of Microbiology, 3-730 BSB, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7982. Fax: (319) 335-9999. E-mail: rebecca-parales{at}uiowa.edu.

dagger Present address: Department of Microbiology, Changwon National University, Kyongnam, South Korea.

Dagger Present address: The Dow Chemical Company, San Diego, Calif.

§ Present address: Lilly Research Laboratories, Indianapolis, Ind.

Journal of Bacteriology, March 2000, p. 1641-1649, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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