Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s

Bühler, Clemens-Jeremias von; Le‐Huu, Priska; Urlacher, Vlada B.

doi:10.1002/cbic.201300271

Cited by 28 publications

(48 citation statements)

References 43 publications

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“…We also provide another way to access the primary (+)‐nootkatone alcohol 3 . Previous studies have shown only allylic hydroxylation products 3 and 26 and the epoxide 25 (Scheme ) . Oxidation of 6 leads even to a greater variety of described products.…”

Section: Resultsmentioning

confidence: 96%

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

et al. 2015

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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.

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Section: Resultsmentioning

confidence: 96%

“…The products identified in this study and the enzymes used are shown in bold. The other derivatization studies of 1 to 3 , 25 , and 26 were conducted by Sowden et al., by Kolev et al., and by von Bühler et al …”

Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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“…Recently we reported on CYP154E1 from Thermobifida fusca YX that accepts a broad range of substrates including geraniol which is converted to 8-hydroxygeraniol [30]. In the present work several further acyclic terpenoids were screened as possible substrates for CYP154E1 and the regio- and chemoselectivity of their oxidation was investigated.…”

Section: Introductionmentioning

confidence: 96%

Selective allylic hydroxylation of acyclic terpenoids by CYP154E1 from Thermobifida fusca YX

Bogazkaya¹,

Bühler²,

Kriening³

et al. 2014

Beilstein J. Org. Chem.

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SummaryAllylic alcohols are valuable precursors in the synthesis of pharmaceutical intermediates, agrochemicals and natural products. Regioselective oxidation of parental alkenes is a challenging task for chemical catalysts and requires several steps including protection and deprotection. Many cytochrome P450 enzymes are known to catalyse selective allylic hydroxylation under mild conditions. Here, we describe CYP154E1 from Thermobifida fusca YX that enables this type of oxidation. Several acyclic terpenoids were tested as possible substrates for CYP154E1, and the regio- and chemoselectivity of their oxidation was investigated. Using a previously established bioinformatics approach we identified position 286 in the active site of CYP154E1 which is putatively involved in substrate binding and thereby might have an effect on enzyme selectivity. To tune regio- and chemoselectivity of the enzyme three mutants at position 286 were constructed and used for substrate oxidation. All formed products were analysed with GC–MS and identified using chemically synthesised authentic samples and known compounds as references. Best regioselectivity towards geraniol and nerol was observed with the wild type enzyme mainly leading to 8-hydroxy derivatives (8-hydroxygeraniol or 8-hydroxynerol) with high selectivity (100% and 96% respectively). Highest selectivities during the oxidation of geranylacetone and nerylacetone were observed with the following variants: V286F led mainly to 7-hydroxygeranylacetone (60% of the total product) and V286A produced predominantly 12-hydroxynerylacetone (75% of total product). Thus, CYP154E1 and its mutants expand the tool-box for allylic hydroxylation in synthetic chemistry.

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“…In fact, Berrie et al purified and characterized this P450 enzyme (24). Very recently, Von et al reported on the substrate screening of two CYP154 enzymes, CYP154E1 of T. fusca and CYP154A8 of N. francinica (25). CYP154E1 and CYP154A8 oxidize many compounds, including saturated fatty acids, primary alcohols, and terpenoids.…”

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confidence: 99%

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Makino

Katsuyama

Otomatsu³

et al. 2014

Appl Environ Microbiol

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c Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio-and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, ⌬ 4 -androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16␣-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio-and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K d ) values of the binding between CYP154C3 and these steroids were less than 8 M as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of ⌬ 4 -androstene-3,17-dione were determined (K m , 31.9 ؎ 9.1 M; k cat , 181 ؎ 4.5 s ؊1 ). We concluded that CYP154C3 is a steroid D-ring 16␣-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16␣-specific hydroxylation activity. C ytochrome P450 monooxygenases (P450s or CYPs) are found in all three domains of life, i.e., archaea, bacteria, and eukarya, and are classified into more than 1,000 families by their amino acid sequence homology (1). They have the ability to hydroxylate inactive carbons of hydrocarbons or aromatic compounds regioand stereospecifically (2, 3). In animals, P450s are mainly involved in xenobiotic metabolism and steroid biosynthesis (4, 5). In plants, many P450s are involved in the biosynthesis of plant hormones and secondary metabolites (6). In contrast, the functions of bacterial P450s are largely unknown, although some bacterial P450s are required for secondary metabolite biosynthesis and assimilation of terpenoids, such as cineol or terpineol (7-10). In spite of their unknown physiological functions, however, bacterial P450s have attracted much attention as a rich resource for new biocatalysts for use in the oxyfunctionalization of chemicals (11, 12). For example, microbial conversion of steroid precursors is widely used for large-scale synthesis of steroid drugs (13-17).CYP154 family members are widely found in actinomycetes, and many putative CYP154 genes have been found in the genomes of Streptomyces species. The structu...

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Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s

Cited by 28 publications

References 43 publications

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

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

Selective allylic hydroxylation of acyclic terpenoids by CYP154E1 from Thermobifida fusca YX

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

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