Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.)

• Published in: Archives of biochemistry and biophysics Vol. 669; pp. 11 - 21
• Main Authors: Lau, Ian C.K., Feyereisen, René, Nelson, David R., Bell, Stephen G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.07.2019
• Subjects: Bacterial metabolism; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 monooxygenases; Escherichia coli - genetics; Ferredoxins; Ferredoxins - genetics; Frankia; Frankia - enzymology; Frankia - genetics; Genes, Bacterial; Phylogeny; Protein Binding; Steroids - metabolism; Terpenes - metabolism; Terpenoids; Cytochrome P450 monooxygenases; Bacterial metabolism; Terpenoids; Frankia; Ferredoxins
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2019.05.007

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as “blooms”. The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe–4S] cluster containing ferredoxins (eight), a single [2Fe–2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria. [Display omitted] •Frankia sp. EuI1c (Frankia inefficax sp.), contains 68 CYP encoding genes.•The CYP189 family (21 members) has undergone gene duplication events classified as a “bloom”.•Genes for eight [3Fe–4S] and one [2Fe–2S] cluster containing ferredoxins were identified.•Three CYP family from Frankia sp. EuI1c, were produced and purified using Escherichia coli.•Substrates including steroids and terpenoids which bind to all three CYPs were identified.