Publication: Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR i from Croton stellatopilosus Ohba
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Issued Date
2015
Resource Type
File Type
application/pdf
ISSN
319422
Other identifier(s)
2-s2.0-84942869318
Rights Holder(s)
Scopus
Bibliographic Citation
Phytochemistry. Vol 118, (2015), p.204-215
Suggested Citation
Sintupachee S., Promden W., Ngamrojanavanich N., Sitthithaworn W., De-Eknamkul W. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR i from Croton stellatopilosus Ohba. Phytochemistry. Vol 118, (2015), p.204-215. doi:10.1016/j.phytochem.2015.08.005 Retrieved from: https://hdl.handle.net/20.500.14740/6088
Abstract
While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase (designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa, respectively. Amino acid sequence comparison showed that both CYP76F45 (63-73%) and CsCPR I (79-83%) share relatively high sequence identities with homologous proteins in other plant species. Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins, showed that only simultaneous incubation of the membrane-bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levels of the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-E-vallesamine, that accumulated in these plant parts. These results suggested that CYP97C27 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus. © 2015 Elsevier Ltd. All rights reserved.
Subject(s)
8-hydroxygeraniol
Complementary DNA
Cytochrome P450
Geraniol
Recombinant protein
Reduced nicotinamide adenine dinucleotide phosphate ferrihemoprotein reductase
Terpene
Amino acid sequence
Croton
Enzymology
Escherichia coli
Genetics
Metabolism
Phylogeny
Plant leaf
Amino Acid Sequence
Croton
Cytochrome P-450 Enzyme System
DNA, Complementary
Escherichia coli
NADPH-Ferrihemoprotein Reductase
Phylogeny
Plant Leaves
Recombinant Proteins
Terpenes
Complementary DNA
Cytochrome P450
Geraniol
Recombinant protein
Reduced nicotinamide adenine dinucleotide phosphate ferrihemoprotein reductase
Terpene
Amino acid sequence
Croton
Enzymology
Escherichia coli
Genetics
Metabolism
Phylogeny
Plant leaf
Amino Acid Sequence
Croton
Cytochrome P-450 Enzyme System
DNA, Complementary
Escherichia coli
NADPH-Ferrihemoprotein Reductase
Phylogeny
Plant Leaves
Recombinant Proteins
Terpenes