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

Abstract

ABSTRACT 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 α 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% (−)-(3 S ,4 R ), respectively] to that formed by wild-type NDO [>98% (+)-(3 R ,4 S )]. 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 -(3 S ,4 R )-dihydrodiol and (−)-phenanthrene cis -(1 S ,2 R )-dihydrodiol from biphenyl and phenanthrene, respectively.