Investigation of cytochromes P450 in myxobacteria: Excavation of cytochromes P450 from the genome of <i>Sorangium cellulosum</i> So ce56

Khatri, Yogan; Hannemann, Frank; Perlova, Olena; Müller, Rolf; Bernhardt, Rita

doi:10.1016/j.febslet.2011.04.035

Cited by 13 publications

(12 citation statements)

References 71 publications

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“…Since cytochromes P450 are external monooxygenases, they require reducing equivalents in the form of electrons for their functional activity, which can be generated either from autologous or heterologous redox partners. Unfortunately, in the genomic context of S. cellulosum So ce56, none of the putative ferredoxin (Fdx) and ferredoxin reductase (FdR) genes are in a cluster with P450s . Nevertheless, we have identified the existence of two different electron transport chains – autologous (Fdx2‐FdR_B and Fdx8‐FdR_B) and heterologous (Adx 4–108 ‐AdR) – for the myxobacterial CYP260A1 and CYP109D1, in which the latter one was found to be more efficient .…”

Section: Resultsmentioning

confidence: 87%

“…Spin‐state shifts upon substrate binding were assayed at room temperature under aerobic conditions using a UV–visible scanning photometer (UV‐2101PC; Shimadzu) equipped with two tandem cuvettes as described previously . The dissociation constants ( K d ) of C10–C13 for CYP267A1 and the L366F mutant were calculated by fitting the peak‐to‐trough difference against the substrate concentration to a nonlinear tight binding quadratic equation as described before .…”

Section: Methodsmentioning

confidence: 99%

“…Since our laboratory has more recently been engaged in the study of cytochromes P450 from the complement (CYPome) of the spore‐forming myxobacterium Sorangium cellulosum So ce56 , we have noticed a naturally occurring heme‐signature variant of CYP267A1, in which the conserved phenylalanine is replaced by leucine (L) in the conserved Cys‐pocket FxxGx(H/R)xCxG. Although the variation LxxGx(H/R)xCxG was also observed in CYP176A (P450cin) from Citrobacter braakii , a detailed biochemical and biophysical characterization has not been performed so far and the implication of this naturally acquired heme‐signature variant was not studied.…”

Section: Introductionmentioning

confidence: 99%

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A natural heme‐signature variant of CYP267A1 from Sorangium cellulosum So ce56 executes diverse ω‐hydroxylation

et al. 2014

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A novel naturally occurring heme-signature variant of CYP267A1 from myxobacterium Sorangium cellulosum So ce56 and its mutant L366F, the actual mimic of the 'conserved' heme-signature of cytochromes P450, were heterologously expressed in Escherichia coli in a soluble form and purified. The UV-visible characteristics of both variants were highly similar. Although leucine replaced the phenylalanine in the heme-signature domain of CYP267A1, EPR measurements of the ligand-free wild-type CYP267A1 and the mutant L366F showed low-spin rhombic species suggesting a conserved heme environment of the P450s. The need of primary redox partners for the orphan P450 was sustained by the bovine redox system and a class-I electron transfer path was provided during fatty acid hydroxylation. CYP267A1 showed higher activity and produced more diverse x-hydroxylated products compared with L366F. In both enzymes the regioselectivity of the fatty acid hydroxylation shifted towards the inner carbon atoms of the fatty acid chains with increasing carbon chain lengths. Our docking results in a homology model of the protein showed that longer fatty acids need to be folded to fit into the binding pocket. In the mutant L366F, the x-1 and x-2 positions which exhibit the largest electron density of the highest occupied molecular orbital are preferred. It is speculated that the leucine heme-signature variant of P450 might have evolved under selective evolutionary pressure, which confers an increased advantage to generate a broader spectrum of related alcohols and carboxylic acids required for the bacterial homeostasis or metabolism in a particular ecological niche.

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Section: Resultsmentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A natural heme‐signature variant of CYP267A1 from Sorangium cellulosum So ce56 executes diverse ω‐hydroxylation

et al. 2014

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“…In the present study, we describe the cloning, expression, and characterization of CYP264B1, one of the 21 P450s of So ce56 in Escherichia coli. Since the gene coding for CYP264B1 (sce8551) is located adjacent upstream of the gene coding for a terpene cyclase (sce8552) with a close distance of 63 base pairs (Khatri et al 2011), it seems that CYP264B1 might be involved in the terpene biosynthesis. The genomic organization of the cluster sce8551-sce8552 is similar to two sesquiterpene biosynthesis gene clusters: one consisting of pentalenene synthase and CYP183A1 from Streptomyces avermitilis (Quaderer et al 2006) and the other one consisting of epiisozizaene synthase and CYP170A1 from Streptomyces coelicolor A3(2) (Zhao et al 2008).…”

Section: Introductionmentioning

confidence: 99%

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase

Khatri

Zapp

et al. 2012

Appl Microbiol Biotechnol

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Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by (1)H and (13)C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the K ( m ) and k (cat) values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.

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“…[41] The increasing number of sequenced bacterial genomes [42] has revealed a large number of putative TS [4][5] and cytochrome P450 genes. [43] The most widespread bacterial TS genes are 2-methylisoborneol (2-MIB) synthase, [44,45] germacradienol/geosmin synthase, [46,47] and epi-isozizaene (EIZ) synthase. [16,18,48] However, the number of functionally characterized TSs has increased only very recently.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

Schifrin

Günnewich

et al. 2014

ChemBioChem

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Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1-geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X-ray diffraction were carried out by employing an Escherichia coli whole-cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)-eremophilene synthase. The very tight binding of (+)-eremophilene (∼0.40 μM), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1-geoA gene cluster is required for the biosynthesis of (+)-eremophilene derivatives.

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

Cited by 13 publications

References 71 publications

A natural heme‐signature variant of CYP267A1 from Sorangium cellulosum So ce56 executes diverse ω‐hydroxylation

A natural heme‐signature variant of CYP267A1 from Sorangium cellulosum So ce56 executes diverse ω‐hydroxylation

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

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