Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: Control by phenylalanine 352 in the alpha subunit

The naphthalene dioxygenase (NDO) system catalyzes the first step in the de gradation of naphthalene by Pseudomonas sp, strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions includ ing cis-dihydroxylation, monooxygenation, and desaturation, Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subuni t of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and p henanthrene, In this study, we replaced Phe-352 with glycine, alanine, isol eucine, threonine, tryptophan, and tyrosine and determined the activity wit h naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F35 2A 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 cis3,4-dihydrodiol [77 and 60% (-)-(3S, 4R), respectively] to t hat formed by wild-type NDO [>98% (+)-(3R,4S)], The F352V mutant enzyme als o formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from p henanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoik uyae B8/36, A recombinant Escherichia coli strain expressing the F352V vari ant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase f rom Pseudomonas putida F1 was used to produce enantiomerically pure (-)-bip henyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.