Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64.

Autor: Taisei Yamamoto, Yoshie Hasegawa, Hiroaki Iwaki
Předmět:
Zdroj: Bioscience, Biotechnology & Biochemistry; Feb2022, Vol. 86 Issue 2, p199-208, 10p
Abstrakt: Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylatedegrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD (P) H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain ( in that order from the N terminus ) . Reverse transcription--polymerase chain reaction results indicated that the P450 CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450 CHC largely preferred NADH (Km = 0.011 m m ) over NADPH (Km = 0.21 mm ). The Kd, Km, and k cat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s-1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450 CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway. [ABSTRACT FROM AUTHOR]
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