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

• Published in: Bioscience, biotechnology, and biochemistry Vol. 86; no. 2; pp. 199 - 208
• Main Authors: Yamamoto, Taisei, Hasegawa, Yoshie, Iwaki, Hiroaki
• Format: Journal Article
• Language: English
• Published: England 24.01.2022
• Subjects: Amino Acid Sequence; Burkholderiaceae - enzymology; Burkholderiaceae - genetics; Cloning, Molecular; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Hydroxylation; Kinetics; NAD - metabolism; NADP - metabolism; Protein Domains; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Paraburkholderia terrae; self-sufficient cytochrome P450; cytochrome P450; cyclohexanecarboxylate; hydroxylase
• ISSN: 1347-6947; 1347-6947
• DOI: 10.1093/bbb/zbab199

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 cyclohexanecarboxylate-degrading 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 P450CHC-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 P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat 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 P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.