Investigation of cytochromes P450 in myxobacteria: Excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

AbstractThe exploitation of cytochromes P450 for novel biotechnological application and for the investigation of their physiological function is of great scientific interest in this post genomic era, where an extraordinary biodiversity of P450 genes has been derived from all forms of life. The study of P450s in the myxobacterium Sorangium cellulosum strain So ce56, the producer of novel secondary metabolites of pharmaceutical interest is the research topic, in which we were engaged since the beginning of its genome sequencing project. We herein disclosed the cytochrome P450 complements (CYPomes) of spore-forming myxobacterial species, Stigmatella aurantiaca DW4/3-1, Haliangium ochraceum DSM 14365 and Myxococcus xanthus DK1622, and their potential pharmaceutical significance has been discussed

Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135350/1/feb212479.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135350/2/feb212479_am.pd

Human P450 CYP17A1: Control of Substrate Preference by Asparagine 202

CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon–carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kd and kcat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme’s native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy–ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s from Sorangium cellulosum So ce56 s

ABSTRACT 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

Das Cytochrom P450 System des Myxcobacteriums Sorangium cellulosum So ce56 und Charakterisierung von CYP109D1 und CYP260A1

This is a very first systematic study of cytochrome P450s from Sorangium cellulosum So ce56. In chapter I, the open reading frames (ORFs) encoding twenty-one cytochrome P450s with nine novel families were identified, and a phylogenetic tree and a physical map were constructed. All P450 ORFs were cloned, expressed in Escherichia coli in a soluble form, purified and characterized. The heterologous redox partners for nine of the P450s were identified. A compound library (~17,000 ligands) was screened with four P450s, and potential substrates and inhibitors were identified. In chapter II, CYP109D1 was shown as a new fatty acid hydroxylase. The GC-MS analysis of TMS-derivatized products of fatty acids showed sub-terminal ω-hydroxylation without any terminal (ω) product. In this chapter, CYP109D1 was also shown as a new biocatalyst for the epoxidation and hydroxylation of monoterpenes like geraniol, nerol and limonene. Furthermore, the selective hydroxylation of sesquiterpenoids, α-ionone and β-ionone to 3-hydroxyα-ionone and 4-hydroxy-β-ionone, confirmed by 1H NMR and 13C NMR, are also shown. The regioselective hydroxylation of the sesquiterpenes was also explained by docking models. In chapter III, steroids (progesterone and DOC) were identified as analogues of potential substrates found by the screening of the compound library with CYP260A1. HPLC analysis of the in vitro products demonstrated the hydroxylation of both the steroids. Moreover, nootkatone, a sesquiterpene, was also shown to be hydroxylated and/or epoxidated by this novel P450.Diese Arbeit ist die bislang erste systematische Untersuchung der Cytochrome P450 aus Sorangium cellulosum So ce56. In Kapitel I wurden die Offenen Leserahmen (ORF) für 21 Cytochrome P450 mit neun neuen Familien identifiziert; ein phylogenetischer Baum und eine physikalische Genkerte wurden erstellt. Alle P450 ORFs wurden kloniert, in löslicher Form in Escherichia coli exprimiert, gereinigt und charakterisiert. Die heterologen Redoxpartner für neun der P450 wurden identifiziert. Eine Bibliothek (~17.000 Liganden) wurde mit vier P450 gescreent und potentielle Substrate und Inhibitoren wurden identifiziert. Kapitel II beschreibt CYP109D1 als neue Fettsäurehydroxylase. GC-MS Analyse der TMS-derivatisierten Produkte der Fettsäuren zeigte ω-Hydroxylierung, aber kein terminales (ω) Produkt. In diesem Kapitel wurde CYP109D1 als ein neuer Biokatalysator für die Epoxidierung und Hydroxylierung von Monoterpenen gezeigt. Zudem wurden die Sesquiterpenoide α-ionon und β-ionon ebenso selektiv zu 3- Hydroxyα-ionon bzw. 4-Hydroxyβ-ionon hydroxyliert, was mittels 1H NMR und 13C NMR Analysen bestätigt werden konnte. Die regioselektive Hydroxylierung der Sesquiterpene konnten auch durch Docking-Modelle erklärt werden. In Kapitel III wurden die Steroide Progesteron und DOC als physiologisch signifikante Analoga der Substrat-Hits für CYP260A1 identifiziert. HPLC Analyse der in vitro Produkte zeigte die Hydroxylierung beider Steroide. Darüber hinaus wurde auch das Sesquiterpen Nootkaton von diesem neuen Cytochrom wurde hydroxyliert und/oder epoxidiert

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

ABSTRACT 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 49-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

Identification of new substrates for the CYP106A1-mediated 11-oxidation and investigation of the reaction mechanism

AbstractCYP106A1 from Bacillus megaterium DSM319 was recently shown to catalyze steroid and terpene hydroxylations. Besides producing hydroxylated steroid metabolites at positions 6β, 7β, 9α and 15β, the enzyme displayed previously unknown 11-oxidase activity towards 11β-hydroxysteroids. Novel examples for 11-oxidation were identified and confirmed by 1H and 13C NMR for prednisolone, dexamethasone and 11β-hydroxyandrostenedione. However, only 11β-hydroxyandrostenedione formed a single 11-keto product. The latter reaction was chosen to investigate the kinetic solvent isotope effect on the steady-state turnover of the CYP106A1-mediated 11-oxidation. Our results reveal a large inverse kinetic isotope effect (∼0.44) suggesting the involvement of the ferric peroxoanion as a reactive intermediate

A Versatile Chemoenzymatic Synthesis for the Discovery of Potent Cryptophycin Analogs

The cryptophycins are a family of macrocyclic depsipeptide natural products that display exceptionally potent antiproliferative activity against drug-resistant cancers. Unique challenges facing the synthesis and derivatization of this complex group of molecules motivated us to investigate a chemoenzymatic synthesis designed to access new analogs for biological evaluation. The cryptophycin thioesterase (CrpTE) and the cryptophycin epoxidase (CrpE) are a versatile set of enzymes that catalyze macrocyclization and epoxidation of over 20 natural cryptophycin metabolites. Thus, we envisioned a drug development strategy involving their use as standalone biocatalysts for production of unnatural derivatives. Herein, we developed a scalable synthesis of 12 new unit A-B-C-D linear chain elongation intermediates containing heterocyclic aromatic groups as alternatives to the native unit A benzyl group

Highly Efficient CYP167A1 (EpoK) dependent Epothilone B Formation and Production of 7-Ketone Epothilone D as a New Epothilone Derivative

Since their discovery in the soil bacterium Sorangium cellulosum, epothilones have emerged as a valuable substance class with promising anti-tumor activity. Because of their benefits in the treatment of cancer and neurodegenerative diseases, epothilones are targets for drug design and pharmaceutical research. The final step of their biosynthesis – a cytochrome P450 mediated epoxidation of epothilone C/D to A/B by CYP167A1 (EpoK) – needs significant improvement, in particular regarding the efficiency of its redox partners. Therefore, we have investigated the ability of various hetero- and homologous redox partners to transfer electrons to EpoK. Hereby, a new hybrid system was established with conversion rates eleven times higher and Vmax of more than seven orders of magnitudes higher as compared with the previously described spinach redox chain. This hybrid system is the most efficient redox chain for EpoK described to date. Furthermore, P450s from So ce56 were identified which are able to convert epothilone D to 14-OH, 21-OH, 26-OH epothilone D and 7-ketone epothilone D. The latter one represents a novel epothilone derivative and is a suitable candidate for pharmacological tests. The results revealed myxobacterial P450s from S. cellulosum So ce56 as promising candidates for protein engineering for biotechnological production of epothilone derivatives