Sesquiterpenoids Produced by Combining Two Sesquiterpene Cyclases with Promiscuous Myxobacterial CYP260B1.

Sesquiterpenes represent a class of important terpenoids with high structural diversity and a wide range of applications. The cyclized core skeletons are generated by sesquiterpene cyclases, and the structural diversity is further increased by a series of modification steps. Cytochromes P450 (P450s) are a class of monooxygenases and one of the main contributors to the structural diversity of natural products. Some of these P450s show a broad substrate range and might be promising candidates for the implementation of cascade reactions. In this study, a combinatorial biosynthesis approach was utilized by the combination of a promiscuous myxobacterial P450 (CYP260B1) with two sesquiterpene cyclases (FgJ01056, FgJ09920) of filamentous fungi. Two oxygenated products, culmorin and culmorone, and a new compound, koraidiol, were successfully generated and characterized. This approach suggests the potential use of noncognate P450s to produce novel oxygenated terpenoids, or to generate a novel biosynthetic route for known terpenoids by a combinatorial biosynthesis strategy.

CYP109E1 from Bacillus megaterium Acts as a 24- and 25-Hydroxylase for Cholesterol.

In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B. megaterium system, yielding about 45 mg L-1 of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol.

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1.

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes.

The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for the enzymatic oxidation of the sesquiterpene alcohols T-muurolol and isodauc-8-en-11-ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro-scale reactions, for the determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450-catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes.

Structure-Based Engineering of Steroidogenic CYP260A1 for Stereo- and Regioselective Hydroxylation of Progesterone.

The production of regio- and stereoselectively hydroxylated steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes hydroxylation of C19 or C21 steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semirational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine. The S276N mutant converted PROG predominantly into 1α-hydroxy-PROG, while the S276I mutant led to 17α-hydroxy-PROG. We solved the high-resolution crystal structures of the PROG-bound S276N and S276I mutants, which revealed two different binding modes of PROG in the active site. The orientations were consistent with the exclusive 1α- (pro-1α binding mode) and 17α-hydroxylation (pro-17α-binding mode) of S276N and S276I, respectively. We observed that water-mediated hydrogen bonds contribute to the stabilization of the polar C3 and C17 substituents of PROG. Both binding modes of PROG may be stabilized in the wild-type enzyme. The change in regioselectivity is mainly driven by destabilizing the alternative binding mode due to steric hindrance and hydrogen bond disruption, caused by the mutations of Ser276. Thus, for the first time, the change in the selectivity of cytochrome P450-mediated steroid hydroxylation created by rational mutagenesis can be explained by the obtained 3D structures of the substrate-bound mutants, providing the basis for further experiments to engineer the biocatalyst toward novel steroid hydroxylation positions.

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

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 ß-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'ß-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-ß-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-ß-damascone, and 3,4-epoxy-ß-damascone). Besides that, a novel compound, 2-hydroxy-ß-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.

CYP260B1 acts as 9α-hydroxylase for 11-deoxycorticosterone.

Steroids and their oxyfunctionalized counterparts are valuable compounds for the pharmaceutical industry; however, the regio- and stereoselective introduction of oxygen is a challenging task for the synthetic chemistry. Thus, cytochromes P450 play an important role for the functionalization of steroidal compounds. In this study, we elucidated the main product of 11-deoxycorticosterone conversion formed by CYP260B1 from Sorangium cellulosum So ce56 as 9α-OH 11-deoxycorticosterone by NMR spectroscopy. This is, to the best of our knowledge, the first identification of a 9α-hydroxylase for this substrate. In addition, the major side product was identified as 21-OH pregna-1,4-diene-3,20-dione. Studies using 1α-OH 11-deoxycorticosterone as substrate suggested that the major side product is formed via dehydrogenation reaction. This side reaction was considerably decreased by employing the CYP260B1-T224A mutant, which showed an increased selectivity of about 75% compared to the 60% of the wild type for the 9α-hydroxylation. To scale up the production, an E. coli based whole-cell system harboring the CYP260B1-T224A variant as well as two heterologous redox partners was used. Employing growing cells in minimal medium led to a productivity of about 0.25g/l/d at a 50ml scale showing the biotechnological potential of this system.

Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1.

Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP260B1 by site-directed mutagenesis to disrupt the substrate positioning and proton delivery, respectively. Furthermore, we used the kinetic solvent isotope effect on the steady-state turnover of the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers.

The guidelines of the Food and Drug Administration and International Conference on Harmonization have highlighted the importance of drug metabolites in clinical trials. As a result, an authentic source for their production is of great interest, both for their potential application as analytical standards and for required toxicological testing. Since we have previously shown promising biotechnological potential of cytochromes P450 from the soil bacterium Sorangium cellulosum So ce56, herein we investigated the CYP267 family and its application for the conversion of commercially available drugs including nonsteroidal anti-inflammatory, antitumor, and antihypotensive drugs. The CYP267 family, especially CYP267B1, revealed the interesting ability to convert a broad range of substrates. We established substrate-dependent extraction protocols and also optimized the reaction conditions for the in vitro experiments and Escherichia coli-based whole-cell bioconversions. We were able to detect activity of CYP267A1 toward seven out of 22 drugs and the ability of CYP267B1 to convert 14 out of 22 drugs. Moderate to high conversions (up to 85% yield) were observed in our established whole-cell system using CYP267B1 and expressing the autologous redox partners, ferredoxin 8 and ferredoxin-NADP(+) reductase B. With our existing setup, we present a system capable of producing reasonable quantities of the human drug metabolites 4'-hydroxydiclofenac, 2-hydroxyibuprofen, and omeprazole sulfone. Due to the great potential of converting a broad range of substrates, wild-type CYP267B1 offers a wide scope for the screening of further substrates, which will draw further attention to future biotechnological usage of CYP267B1 from S. cellulosum So ce56.

Selective oxidation of carotenoid-derived aroma compounds by CYP260B1 and CYP267B1 from Sorangium cellulosum So ce56.

Due to their bioactive properties as well as their application as precursors in chemical synthesis, hydroxylated isoprenoids and norisoprenoids are very valuable compounds. The efficient hydroxylation of such compounds remains a challenge in organic chemistry caused by the formation of a variety of side products and lack of overall regio- and stereoselectivity. In contrast, cytochromes P450 are known for their selective oxidation under mild conditions. Here, we demonstrate for the first time the ability of myxobacterial CYP260B1 and CYP267B1 from Sorangium cellulosum So ce56 to oxidize such carotenoid-derived aroma compounds. A focused library of 14 substrates such as ionones, damascones, as well as some of their isomers and derivatives was screened in vitro. Both P450s were capable of an efficient oxidation of all tested compounds. CYP260B1-dependent conversions mainly formed multiple products, whereas conversions by CYP267B1 resulted predominantly in a single product. To identify the main products by NMR spectroscopy, an Escherichia coli-based whole-cell system was used. CYP267B1 showed a hydroxylase activity towards the formation of allylic alcohols. Likewise, CYP260B1 performed the allylic hydroxylation of ß-damascone [(E)-1-(2,6,6-trimethylcyclohex-1-enyl)but-2-en-1-one] and δ-damascone [(E)-1-(2,6,6-trimethylcyclohex-3-enyl)but-2-en-1-one]. Moreover, CYP260B1 showed an epoxidase activity towards ß-ionone [(E)-4-(2,6,6-trimethylcyclohex-1-enyl)but-3-en-2-one] as well as the methyl-substituted α-ionone derivatives raldeine [(E)-1-(2,6,6-trimethylcyclohex-2-enyl)pent-1-en-3-one] and isoraldeine [(E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-enyl)but-3-en-2-one]. In addition, to known products, also novel products such as 2-OH-δ-damascone [(E)-1-(5-hydroxy-2,6,6-trimethylcyclohex-3-enyl)but-2-en-1-one], 3-OH-allyl-α-ionone [(E)-1-(4-hydroxy-2,6,6-trimethylcyclohex-2-enyl)hepta-1,6-dien-3-one], and 4-OH-allyl-ß-ionone [(E)-1-(3-hydroxy-2,6,6-trimethylcyclohex-1-enyl)hepta-1,6-dien-3-one] were identified during our studies.

New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56.

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non-activated C-H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)-nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.

Conversions of tricyclic antidepressants and antipsychotics with selected P450s from Sorangium cellulosum So ce56.

Human cytochromes P450 (P450s) play a major role in the biotransformation of drugs. The generated metabolites are important for pharmaceutical, medical, and biotechnological applications and can be used for derivatization or toxicological studies. The availability of human drug metabolites is restricted and alternative ways of production are requested. For this, microbial P450s turned out to be a useful tool for the conversion of drugs and related derivatives. Here, we used 10 P450s from the myxobacterium Sorangium cellulosum So ce56, which have been cloned, expressed, and purified. The P450s were investigated concerning the conversion of the antidepressant drugs amitriptyline, clomipramine, imipramine, and promethazine; the antipsychotic drugs carbamazepine, chlorpromazine, and thioridazine, as well as their precursors, iminodibenzyl and phenothiazine. Amitriptyline, chlorpromazine, clomipramine, imipramine, and thioridazine are efficiently converted during the in vitro reaction and were chosen to upscale the production by an Escherichia coli-based whole-cell bioconversion system. Two different approaches, a whole-cell system using M9CA medium and a system using resting cells in buffer, were used for the production of sufficient amounts of metabolites for NMR analysis. Amitriptyline, clomipramine, and imipramine are converted to the corresponding 10-hydroxylated products, whereas the conversion of chlorpromazine and thioridazine leads to a sulfoxidation in position 5. It is shown for the first time that myxobacterial P450s are efficient to produce known human drug metabolites in a milligram scale, revealing their ability to synthesize pharmaceutically important compounds.