Specific N‐demethylation of verapamil by cytochrome P450 from Streptomyces griseus ATCC 13273

Norverapamil, the N‐demethylated derivative of verapamil, is a novel promising leading compound for attenuating multidrug resistance with less side effects compared with verapamil. However, the efficient synthetic method for norverapamil is absent. In this study, an innovative biotechnological method based on enzymatic catalysis was presented for the high‐efficient production of norverapamil. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, was identified to carry out a one‐step specific N‐demethylation of verapamil along with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) as the redox partner. Docking calculations rationalized the specific N‐demethylation observed in experiment and identified important amino acid residues for verapamil binding. Furthermore, a CYP105D1‐based whole‐cell system in E. coli BL21(DE3) was established and optimized for highly efficient N‐demethylation of verapamil. The bioconversion rate of verapamil by the whole cell system came up to 60.16% within 24 hours under the optimized conditions. These results demonstrated the high potential of CYP105D1‐based biocatalytic system for norverapamil production.     

Screening of bacterial cytochrome P450s responsible for regiospecific hydroxylation of (iso)flavonoids

Screening of cytochrome P450 monoxygenases responsible for the regiospecific hydroxylation of flavones, isoflavones and chalcones was attempted using a P450 library constructed from Streptomyces avermitilis MA4680, Bacillus and Nocardia farcinica IFM10152 strains. As electron transfer redox partners with the P450s in Escherichia coli system, putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida were used. Among the 50 soluble P450s in the library screened, three cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 from S. avermitilis MA4680 showed good hydroxylation activities towards flavones and isoflavones. However, low product yields prevented us from identifying complete structure of the products. By using S. avermitilis MA4680 as their expression host, further analysis identified that CYP107Y1(SAV2377), CYP125A2(SAV5841) and CYP107P2(SAV4539) showed good regiospecific hydroxylation activities towards genistein (4',5,7-trihydroxyisoflavone), chrysin (5,7-dihydroxyisoflavone) and apigenin (4',5,7-dihydroxyisoflavone) to produce 3',4',5,7,-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydroxyflavone and 3',4',5,7,-tetrahydroxyflavone, respectively. Analyses of the reaction products were performed using HPLC, ESI-MS-MS and GC-MS and 1H NMR.     

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K_m and k_cat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min^?1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO‐difference spectra at 450 nm using the whole cell extract. When the whole‐cell reaction for the 3′‐hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6‐fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3′,4′‐trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h. Biotechnol. Bioeng. 2010. 105: 697–704. © 2009 Wiley Periodicals.     

Cytochrome P450 enzymes from the metabolically diverse bacterium Rhodopseudomonas palustris

Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.     

Protein engineering of CYP105s for their industrial uses

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D₃ 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Effective cytochrome P450 (CYP) inhibitor isolated from thyme (Thymus saturoides) purchased from a Japanese market

A highly polymethylated flavone that effectively inhibited cytochrome P450s (CYPs) 1A2 and 3A4 (IC(50) = 2.41 and 1.71 μM) in vitro was isolated from thyme leaves (Thymus saturoides) purchased from a Japanese market. Its structure was spectroscopically identified as 4',5-dihydroxy-3',6,7,8-tetramethoxy flavone (8-methoxycirsilineol, 1). This is the first report describing a strong inhibitor of CYP1A2 and 3A4 isolated from Thymus saturoides.     

Steady-state kinetic investigation of cytochrome P450cam: interaction with redox partners and reaction with molecular oxygen

Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.     

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.     

Designing a whole-cell biotransformation system in <Emphasis Type="Italic">Escherichia coli</Emphasis> using cytochrome P450 from <Emphasis Type="Italic">Streptomyces peucetius</Emphasis>

A biotransformation system was designed to co-express CYP107P3 (CSP4), cytochrome P450, from Streptomyces peuceticus, along with CamA (putidaredoxin reductase) and CamB (putidaredoxin) from Pseudomonas putida, the necessary reducing equivalents, in a class I type electron-transfer system in E. coli BL21 (DE3). This was carried out using two plasmids with different selection markers and compatible origins of replication. The study results showed that this biotransformation system was able to mediate the O-dealkylation of 7-ethoxycumarin.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (k_cat = 39–91 s⁻¹) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity.     

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.     

Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards beta-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 degrees C. In this system, beta-carotene was hydroxylated to beta-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low beta-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated beta-carotene at 3- and also 3'-positions, resulting in beta-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol beta-cryptoxanthin produced per nmol P450 per min.     

FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple

Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.     

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.     

Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes

An approach to measure the activity of single oligomers of the heme-containing enzyme cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine the integral and local Young’s modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively.     

Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones

Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.     

The active site of the thermophilic CYP119 from Sulfolobus solfataricus

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.     

Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts

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.