Substrate Binding to 15β-Hydroxylase (CYP106A2) Probed by FT Infrared Spectroscopic Studies of the Iron Ligand CO Stretch Vibration

Simgen, Birgit; Contzen, Jörg; Schwarzer, Rolf; Bernhardt, Rita; Jung, Christiane

doi:10.1006/bbrc.2000.2348

Cited by 56 publications

(36 citation statements)

References 23 publications

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“…Evaluation of the levels of correctly folded recombinant P450 was carried out by determining the CO-reduced difference spectra (9,40). The cells were grown in Terrific broth alone or with the heme precursor, ALA, or with ALA and FeCl 3.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

Hussain

Ward

2003

Appl Environ Microbiol

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The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.

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Section: Resultsmentioning

confidence: 99%

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

Hussain

Ward

2003

Appl Environ Microbiol

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“…Interestingly, abietic acid-like the steroidal substrates of CYP106A2 [21] -did not induce a type I spectrum during binding. This property had been observed with CYP106A2 and the substrate deoxycorticosterone and afterwards was also noticed for the binding of substrates to other P450s.…”

Section: Substrate Predictionmentioning

confidence: 92%

“…Expression and purification of CYP106A2 were performed as described in the literature. [12,21] The protein concentration was calculated with use of E 414 = 9.8 mm À1 cm À1 for adrenodoxin , [29] E 450 = 11.3 mm À1 cm À1 for adrenodoxin reductase, [30] and E 417 = 91 mm À1 cm À1 for CYP106A2. [12] Automated library screening: A library consisting of 16 671 small molecules was screened.…”

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confidence: 99%

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

et al. 2011

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The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3-oxo-Δ(4) -steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15β-position. We combined a high-throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the V(max) and K(m) values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12α- and 12β-hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole-cell catalyst for the selective allylic 12α- and 12β-hydroxylation was applied to produce the hydroxylated products.

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“…[66] Bacterial expression and purification of recombinant CYP106A2 and mutants: The CYP106A2 cDNA was a kind gift from Dr. Raimund Rauschenbach (Schering AG, Berlin, Germany). Expression of CYP106A2 was performed as described in Simgen et al [67] Purification was performed according to Lisurek et al [6] Site directed mutagenesis: Mutagenesis of residues S394, A395, T396, G397 and Q398 was accomplished according to the protocol of the Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) by using oligonucleotide primers that were synthesised by MWG Biotech AG (Ebersberg, Germany) and the plasmid CYP106A2/pKKHC (NcoI/HindIII). [68] The three mutants I86T, E90V/ D185G and K27R/I71T/I215T were obtained by using a modified plasmid CYP106A2/pKKHC (KpnI/SalIII).…”

Section: Methodsmentioning

confidence: 99%

Theoretical and Experimental Evaluation of a CYP106A2 Low Homology Model and Production of Mutants with Changed Activity and Selectivity of Hydroxylation

et al. 2008

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Steroids are important pharmaceutically active compounds. In contrast to the liver drug-metabolising cytochrome P450s, which metabolise a variety of substrates, steroid hydroxylases generally display a rather narrow substrate specificity. It is therefore a challenging goal to change their regio- and stereoselectivity. CYP106A2 is one of only a few bacterial steroid hydroxylases and hydroxylates 3-oxo-Delta4-steroids mainly in 15beta-position. In order to gain insights into the structure and function of this enzyme, whose crystal structure is unknown, a homology model has been created. The substrate progesterone was then docked into the active site to predict which residues might affect substrate binding. The model was substantiated by using a combination of theoretical and experimental investigations. First, numerous computational structure evaluation tools assessed the plausibility of its protein geometry and its quality. Second, the model explains many key properties of common cytochrome P450s. Third, two sets of mutants have been heterologously expressed, and the influence of the mutations on the catalytic activity towards deoxycorticosterone and progesterone has been studied experimentally: the first set comprises six mutations located in the structurally variable regions of this enzyme that are very difficult to predict by cytochrome P450 modelling (K27R, I86T, E90V, I71T, D185G and I215T). For these positions, no participation in the active-site formation was predicted, or could be experimentally demonstrated. The second set comprises five mutants in substrate recognition site 6 (S394I, A395L, T396R, G397P and Q398S). For these residues, participation in active-site formation and an influence on substrate binding was predicted by docking. These mutants are based on an alignment with human CYP11B1, and in fact most of these mutants altered the active-site structure and the hydroxylation activity of CYP106A2 dramatically.

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Substrate Binding to 15β-Hydroxylase (CYP106A2) Probed by FT Infrared Spectroscopic Studies of the Iron Ligand CO Stretch Vibration

Cited by 56 publications

References 23 publications

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

Theoretical and Experimental Evaluation of a CYP106A2 Low Homology Model and Production of Mutants with Changed Activity and Selectivity of Hydroxylation

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