CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium

Abstract

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'β-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

NarL, a Novel Repressor for CYP108j1 Expression during PAHs Degradation in Rhodococcus sp. P14

“…CYP108D1 from Novosphingobium aromaticivorans DSM12444 was reported to have the activity in the oxidation of polycyclic aromatic hydrocarbons, such as phenanthrene, biphenyl, and phenylcyclohexane [30]. CYP108A1 can effectively hydroxylate the terpene for α-terpineol oxidation [31]. CYP108N7 from Rhodococcus NBRC 100605 is also able to catalyze the epoxidation, hydroxylation, demethylation, and dehalogenation of low molecular weight PAHs and their products [32].…”

Characterization and structure‐guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

“…Studies on the steroid hydroxylating CYP109E1 from B. megaterium (36% identity to CYP109Q5) showed that this enzyme is more closely related to the steroid hydroxylase CYP106A1 in comparison with the already characterized CYP109 enzymes (J o zwik et al, 2016). Classification into a P450 family thus represents a strong evidence for a particular substrate spectrum; this is reflected by CYP109B1 and CYP109E1 that also functionalize a broad spectrum of substrates including steroids, fatty acids and terpenes (Girhard et al, 2010;Putkaradze et al, 2017). However, the preferences or natural substrates may differ in large and heterogeneous P450 families such as the CYP109 family.…”

Glycosylation of bioactive C13-apocarotenols inNicotiana benthamianaandMentha × piperita

“…It has recently been shown that bacterial cytochrome P450s, among them the hydroxylase CYP109E1 from Bacillus megaterium, can oxidize various apocarotenoids (Khatri et al 2010;Litzenburger and Bernhardt 2016) including ionones (Putkaradze et al 2017). Therefore, B. megaterium expressing CYP109E1…”

“…In addition, 3-hydroxy-α-ionol, 4-hydroxy-β-ionol, 3-hydroxy-α-ionone, 4-hydroxy-βionone, 3-hydroxy-α-damascone and 4-hydroxy-β-damascone were tested, which were produced by P450-catalyzed biotransformation using Bacillus megaterium (Putkaradze et al 2017). For the substrate screening, 50 µl crude protein extract, 100…”

“…megaterium whole-cell system was applied (Putkaradze et al 2017). C 13apocarotenoid (α-or β-ionol, αor β-ionone, αor β-damascone) was added to the cell-suspension with a final concentration of 40 mg L -1 .…”