title: Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.
abstract: The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.
authors.affiliation: Department of Microbiology, The University of Iowa, Iowa City, 52242, USA.
authors.forename: H
authors.initials: H
authors.lastname: Jiang
authors.forename: R E
authors.initials: RE
authors.lastname: Parales
authors.forename: N A
authors.initials: NA
authors.lastname: Lynch
authors.forename: D T
authors.initials: DT
authors.lastname: Gibson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000737:chemistry*
chemical_list: D007501:Iron; D010105:Oxygenases; C023172:toluene dioxygenase