Open AccessIdentification of core active site residues of the sulfur oxygenase reductase from Acidianus ambivalens by site‐directed mutagenesis
Author(s) -
Urich Tim,
Kroke Anja,
Bauer Christian,
Seyfarth Kerstin,
Reuff Muriel,
Kletzin Arnulf
Publication year - 2005
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/j.femsle.2005.05.031
Subject(s) - active site , oxygenase , biochemistry , chemistry , alanine , mutagenesis , enzyme , mutant , site directed mutagenesis , heme oxygenase , reductase , wild type , heme , stereochemistry , amino acid , gene
The sulfur oxygenase reductase (SOR) is the initial enzyme in the sulfur oxidation pathway of Acidianus ambivalens. The SOR is composed of 308 aa residues, three of which are cysteines, and contains a mononuclear non‐heme iron site. Mutations of the suspected iron‐binding residues H 86 , H 90 and E 114 to alanine resulted in inactive enzyme with no iron incorporated, whereas an E 114 D mutant showed 1% of wild type activity. The mutation of C 31 to alanine and serine caused inactivity of the enzyme, however, the iron content was the same as in the wild type. C 101 A, C 104 S/A, and C 101/104 S/A double mutants caused a decrease in specific activity to 10–43% of the wild type while the C 101 S mutant showed only 1% activity of the wild type. The drop in activity of the C 101 S and E 114 D mutants was accompanied with a proportional decrease in iron content. In all cases the oxygenase and reductase partial reactions were equally affected. It was concluded that the Fe site with H 86 , H 90 and E 114 as ligands and C 31 constitute the core active site whereas C 101 and C 104 optimize reaction conditions.