Premium
Biochemical characterization of an N‐terminally histidine tagged styrene oxide isomerase from Pseudonomas putida (S12)
Author(s) -
Liao Sindy,
Gassner George
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.928.5
Subject(s) - styrene oxide , styrene , chemistry , isomerase , isomerization , substrate (aquarium) , phenylacetaldehyde , enzyme , biochemistry , copolymer , catalysis , organic chemistry , biology , polymer , ecology
In the first step of the styrene catabolic and detoxification pathway styrene is transformed to styrene oxide, a biochemical alkylating agent and carcinogen. In Pseudomonas putida (S12), Styrene Oxide Isomerase (SOI) performs the critical role of catalyzing the isomerization of styrene oxide to phenylacetaldehyde, a less toxic intermediate. The detailed kinetic mechanism of SOI is a subject of current investigation. Conditions have been identified that allow an N‐terminally histidine‐tagged version of Styrene Oxide Isomerase (N‐SOI) to be over‐expressed and partially purified by Ni 2+ ‐affinity chromatography in a catalytically active form for biochemical characterization. An enzyme‐coupled assay with phenylacetaldehyde dehydrogenase was developed to indirectly monitor the activity of N‐SOI and kinetically characterize the enzyme by using a microplate reader. By using this assay steady‐state kinetic parameters of N‐SOI with (S)‐styrene oxide were determined to be Vmax = (4.6 ± 0.5) uM/min and K M = (1.6 ± 0.7) uM at pH 8 and 25°C. The substrate specificity and catalytic role of protein‐protein interactions of N‐SOI with the other components of the styrene degradation pathway will be reported.