Open AccessHeterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex
Author(s) -
Champreda Verawat,
Zhou NingYi,
Leak David J.
Publication year - 2004
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.2004.09.002
Subject(s) - ferredoxin , monooxygenase , maltose binding protein , chemistry , reductase , oxidoreductase , oxygenase , biochemistry , stereochemistry , biology , fusion protein , recombinant dna , enzyme , gene , cytochrome p450
The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N‐terminal affinity tagged fusion and native forms in Escherichia coli . However, attempts to express the NADH‐oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins. Nevertheless, the recombinant reductase from the toluene 4‐monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system. In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady‐state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction. The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25–0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two‐fold symmetry binding sites on the oxygenase.