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Journal of Bacteriology, October 2000, p. 5495-5504, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene cis-Dihydroxylation: Control by Phenylalanine 352 in the alpha  Subunit

Rebecca E. Parales,1,* Sol M. Resnick,1,dagger Chi-Li Yu,1 Derek R. Boyd,2 Narain D. Sharma,2 and David T. Gibson1

Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242,1 and School of Chemistry, The Queen's University of Belfast, Belfast, BT9 5AG, United Kingdom2

Received 8 March 2000/Accepted 28 June 2000

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the alpha  subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (-)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.

* 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: The Dow Chemical Company, San Diego, Calif.

Journal of Bacteriology, October 2000, p. 5495-5504, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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