Martin Litzenburger

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.

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 β-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 β-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-β-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.

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.

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.

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.

CYP260B1 acts as 9α-hydroxylase for 11-deoxycorticosterone

HIGHLIGHTSCYP260B1 acts as 9&agr;‐hydroxylase for 11‐deoxycorticosterone.The T224A mutant of CYP260B1 increases the selectivity.A productivity of about 0.25 g/l/d can be achieved. ABSTRACT 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 9&agr;‐OH 11‐deoxycorticosterone by NMR spectroscopy. This is, to the best of our knowledge, the first identification of a 9&agr;‐hydroxylase for this substrate. In addition, the major side product was identified as 21‐OH pregna‐1,4‐diene‐3,20‐dione. Studies using 1&agr;‐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 9&agr;‐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 0.25 g/l/d at a 50 ml 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.