A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans.
Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized beta-ionone to predominantly 3-R-hydroxy-beta-ionone and 4-hydroxy-beta-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L(-1) of 5-exo-hydroxycamphor at rates of up to 64 microM (gram of cell dry weight)(-1) min(-1). These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.
Bell
Stephen G
SG
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX13QR, UK. stephen.bell@chem.ox.ac.uk
Dale
Alison
A
Rees
Nicholas H
NH
Wong
Luet-Lok
LL
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D002164:Camphor / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D045792:Norisoprenoids / Q000378:metabolism; D010084:Oxidation-Reduction; D042301:Sphingomonadaceae / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity
C081714:2Fe-2S ferredoxin; D001426:Bacterial Proteins; D005288:Ferredoxins; D045792:Norisoprenoids; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; C008157:beta-ionone