Putidaredoxin Reductase-Putidaredoxin-Cytochrome P450cam Triple Fusion Protein

Fusion proteins of cytochrome P450cam with putidaredoxin (Pd) and putidaredoxin reductase (PdR), the two proteins required to transfer electrons from NADH to P450cam, were constructed by fusing cDNAs encoding the three proteins in the expression vector pCWori+. Several fusion proteins, in which the order of the three protein domains and the linkers between them were varied, were expressed in Escherichia coli, purified, and characterized. The highest activity (kcat = 30 min-1) was obtained with a PdR-Pd-P450cam construct in which the peptides TDGTASS and PLEL were used, respectively, to link the PdR to the Pd and the Pd to the P450cam domains. Oxygen and NADH consumption is tightly coupled to substrate oxidation in the fusion proteins. The rate-limiting step in the catalytic turnover of these fusion proteins is electron transfer from Pd to P450cam. This is indicated by high rates of electron transfer from the PdR and Pd domains to exogenous electron acceptors, by an increase in the activity of the P450cam domain upon addition of exogenous Pd, and by the high activity of wild-type P450cam when incubated with a PdR-Pd fusion protein. E. coli cells expressing the PdR-Pd-P450cam fusion protein efficiently oxidize camphor to 5-exo-hydroxycamphor and 5-oxocamphor. E. coli cells expressing the triple fusion protein thus constitute the first heterologous self-sufficient catalytic system for the oxidation of camphor and other substrates by P450cam. Fusion proteins of cytochrome P450cam with putidaredoxin (Pd) and putidaredoxin reductase (PdR), the two proteins required to transfer electrons from NADH to P450cam, were constructed by fusing cDNAs encoding the three proteins in the expression vector pCWori+. Several fusion proteins, in which the order of the three protein domains and the linkers between them were varied, were expressed in Escherichia coli, purified, and characterized. The highest activity (kcat = 30 min-1) was obtained with a PdR-Pd-P450cam construct in which the peptides TDGTASS and PLEL were used, respectively, to link the PdR to the Pd and the Pd to the P450cam domains. Oxygen and NADH consumption is tightly coupled to substrate oxidation in the fusion proteins. The rate-limiting step in the catalytic turnover of these fusion proteins is electron transfer from Pd to P450cam. This is indicated by high rates of electron transfer from the PdR and Pd domains to exogenous electron acceptors, by an increase in the activity of the P450cam domain upon addition of exogenous Pd, and by the high activity of wild-type P450cam when incubated with a PdR-Pd fusion protein. E. coli cells expressing the PdR-Pd-P450cam fusion protein efficiently oxidize camphor to 5-exo-hydroxycamphor and 5-oxocamphor. E. coli cells expressing the triple fusion protein thus constitute the first heterologous self-sufficient catalytic system for the oxidation of camphor and other substrates by P450cam.