TY - JOUR
T1 - Intramolecular electron transfer in a cytochrome P450cam system with a site-specific branched structure
AU - Hirakawa, Hidehiko
AU - Kamiya, Noriho
AU - Tanaka, Tsutomu
AU - Nagamune, Teruyuki
N1 - Funding Information:
We thank Prof Goto and Dr Ichinose of Kyushu University for plasmids encoding the genes for putidaredoxin and putidaredoxin reductase and Prof Sligar of University of Illinois for a plasmid encoding the gene for P450cam. We are also grateful to Ajinomoto Co., Inc., for providing the TGase sample. This work was partly supported by the 21st century COE program ‘Human-Friendly Material Based on Chemistry’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2007/9
Y1 - 2007/9
N2 - Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min-1) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.
AB - Cytochrome P450 (P450) is an attractive oxygenase due to the diverse catalytic reactions and the broad substrate specificity. Class I P450s require an excess concentration (more than 10 times) of iron-sulfur proteins, which transfer electrons to P450s, to attain the maximum catalytic activity and this requirement is a critical bottleneck for practical applications. Here, we show a site-specific branched fusion protein of P450 with its electron transfer proteins using enzymatic cross-linking with transglutaminase. A branched fusion protein of P450 from Pseudomonas putida (P450cam), which was composed of one molecule each of P450cam, putidaredoxin (Pdx) and Pdx reductase, showed higher catalytic activity (306 min-1) and coupling efficiency (99%) than the equimolar reconstitution system due to the intramolecular electron transfer. The unique site-specific branched structure simply increased local concentration of proteins without denaturation of each protein. Therefore, enzymatic post-translational protein manipulation can be a powerful alternative to conventional strategies for the creation of multicomponent enzyme systems with novel proteinaceous architecture.
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U2 - 10.1093/protein/gzm045
DO - 10.1093/protein/gzm045
M3 - Article
C2 - 17827502
AN - SCOPUS:35348837263
SN - 1741-0126
VL - 20
SP - 453
EP - 459
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 9
ER -