Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents.
The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51 degrees C, and the half-life at 50 degrees C was with 100 min more than ten times longer than that of CYP102A1 (8 min).
Eiben
Sabine
S
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Bartelmäs
Heike
H
Urlacher
Vlada B
VB
D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C445019:CYP102A3 protein, Bacillus subtilis; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases