Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli.
We tested the synthesis and in vivo function of the inducible alkane hydroxylase of Pseudomonas oleovorans GPo1 in several Escherichia coli recombinants. The enzyme components (AlkB, AlkG and AlkT) were synthesized at various rates in different E. coli hosts, which after induction produced between twofold and tenfold more of the Alk components than did P. oleovorans. The enzyme components were less stable in recombinant E. coli hosts than in P. oleovorans. In addition, the specific activity of the alkane mono-oxygenase component AlkB was five or six times lower in E. coli than in P. oleovorans. Evidently, optimal functioning of the hydroxylase system requires factors or a molecular environment that are available in Pseudomonas but not in E. coli. These factors are likely to include correct interactions of AlkB with the membrane and incorporation of iron into the AlkG and AlkB apoproteins.
Staijen
I E
IE
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, Switzerland.
Van Beilen
J B
JB
Witholt
B
B
D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A