Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity

The importance of five amino acids at the active site of the multicomponent naphthalene, dioxygenase (NDO) system was determined by generating site-di rected mutations in various combinations. The substrate specificities of th e mutant enzymes were tested with the substrates indole, indoline, 2-nitrot oluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of th ese substrates measured the ability of the mutant enzymes to catalyze dioxy genation, monooxygenation, and desaturation reactions. In addition, the pos ition of oxidation and the enantiomeric composition of products were charac terized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also cat alyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at positio n. 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed f rom naphthalene, but in the presence of the F3521 mutation, changes at posi tions 206 and 295 also affected enantioselectivity. Major shifts in regiose lectivity with biphenyl and phenanthrene resulted with several of the singl y, doubly, and triply mutated enzymes. A new product not formed by the wild -type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major prod uct from phenanthrene by enzymes With two (A206I/F3521) or three amino acid substitutions (A2061/ F3521/H2951). The results, indicate that a variety o f amino acid substitutions are tolerated at the active site of NDO.