Open AccessSubstrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme
Author(s) -
Rebecca E. Parales,
Kyung Ho Lee,
Sol M. Resnick,
Haiyan Jiang,
Daniel J. Lessner,
David T. Gibson
Publication year - 2000
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.182.6.1641-1649.2000
Subject(s) - active site , dioxygenase , stereochemistry , naphthalene , enzyme , amino acid , alanine , phenanthrene , biphenyl , substrate (aquarium) , biochemistry , chemistry , biology , organic chemistry , ecology
The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzescis -dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation ofcis -naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R ,2S ], compared 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 to the active-site iron, resulted in a completely inactive enzyme.