Substrate specificity of naphthalene dioxygenase: Effect of specific aminoacids at the active site of the enzyme

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp , strain NCIB 981.6-4, The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydroph obic, which is consistent with the enzyme's preference for aromatic hydroca rbon substrates, Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective, Site-directed mut agenesis was used to determine the contributions of several active-site res idues to these aspects of catalysis. Amino acid substitutions at Asn-201, P he-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no ef fect on product formation with naphthalene or biphenyl as substrates and ha d slight but significant effects on product formation from phenanthrene, Am ino acid substitutions at Phe-352 resulted in the formation of cis-naphthal ene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compare d 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 t o the active-site iron, resulted in a completely inactive enzyme.