Publication:
Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba

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Issued Date

2014

Resource Type

Article

File Type

application/pdf

ISSN

1689452

DOI

10.1016/j.plantsci.2014.09.001

Other identifier(s)

2-s2.0-84907463649

Rights Holder(s)

Scopus

Bibliographic Citation

Plant Science. Vol 229, (2014), p.131-141

Suggested Citation

Sintupachee S., Ngamrojanavanich N., Sitthithaworn W., De-Eknamkul W. Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba. Plant Science. Vol 229, (2014), p.131-141. doi:10.1016/j.plantsci.2014.09.001 Retrieved from: https://hdl.handle.net/20.500.14740/6893

Title

Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba

Author(s)

Sintupachee S.
 Ngamrojanavanich N.
 Sitthithaworn W.
 De-Eknamkul W.

Abstract

The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79. kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85-93%) and CPR I (79-83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97. C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus, the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway. © 2014 The Authors.

Subject(s)

Cytochrome P450
 Fatty alcohol
 Messenger RNA
 Plaunotol
 Recombinant protein
 Reduced nicotinamide adenine dinucleotide phosphate ferrihemoprotein reductase
 Amino acid sequence
 Biosynthesis
 Chemistry
 Croton
 Enzymology
 Escherichia coli
 Gene expression profiling
 Gene expression regulation
 Genetics
 Metabolism
 Molecular cloning
 Molecular genetics
 Phylogeny
 Sequence alignment
 Thin layer chromatography
 Amino Acid Sequence
 Biosynthetic Pathways
 Chromatography, Thin Layer
 Cloning, Molecular
 Croton
 Cytochrome P-450 Enzyme System
 Escherichia coli
 Fatty Alcohols
 Gene Expression Profiling
 Gene Expression Regulation, Enzymologic
 Gene Expression Regulation, Plant
 Molecular Sequence Data
 NADPH-Ferrihemoprotein Reductase
 Phylogeny
 Recombinant Proteins
 RNA, Messenger
 Sequence Alignment

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URI

https://hdl.handle.net/20.500.14740/6893

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Scopus: Year 1983-2021

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