Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.     

Cofactor recycling with immobilized heterologous cytochrome P450 105D1 (CYP105D1)

Immobilisation of cells and enzymes can be a convenient and rapid way for testing and transforming substances. Cytochromes P450 may be useful in numerous biotransformations of varied lipophilic substrates, performing both regio- and stereo-specific monooxygenation reactions. However, one limitation of their use in vitro is the requirement of cofactor for the supply of electrons in the catalytic cycle. Here we report CYP105D1 from Streptomyces griseus expressed in Escherichia coli can be immobilised from cell-free extracts using DE52, that the immobilised protein is active in bioconversions and that a requirement for cofactor can be sustained by a recycling system for NADH regeneration.     

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.     

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1′, respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B′ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.     

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis: Conformational Flexibility and Histidine Ligation State

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1′-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate.     

Cytochrome P450 1A1 (CYP1A1) in blood lymphocytes evidence for catalytic activity and mRNA expression.

Studies initiated to characterise the catalytic activity and expression of CYP1A1 in rat blood lymphocytes revealed significant activity of 7-ethoxyresorufin-O-deethylase (EROD) in rat blood lymphocytes. Pretreatment with 3-methylcholanthrene (MC) and beta-naphthoflavone (NF) resulted in significant induction in the activity of lymphocyte EROD suggesting that like the liver enzyme, EROD activity in lymphocytes is inducible and is mediated by the MC inducible isoenzymes of P450. The increase in the activity of EROD was associated with a significant increase in the apparent Vmax and affinity of the substrate towards EROD. That this increase in the activity of EROD could be primarily due to the increase in the expression of CYP1A1 isoenzymes was demonstrated by RT-PCR and western immunoblotting studies indicating an increase in the expression of CYP1A1 in blood lymphocytes after MC pretreatment. Significant inhibition in the EROD activity of MC induced lymphocyte by anti-CYP1A1/1A2 and alpha-naphthoflavone further provided evidence that the CYP1A1/1A2 isoenzymes are involved in the activity of EROD in blood lymphocytes. The data indicating similarities in the regulation of CYP1A1 in blood lymphocytes with the liver isoenzyme suggests that factors which may affect expression of CYP1A1 in liver may also affect expression in blood lymphocytes and that blood lymphocytes could be used as a surrogates for studying hepatic expression of the xenobiotic metabolising enzymes.     

Biotransformation of flavone by CYP105P2 from Streptomyces peucetius

Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LCMS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS. A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.     

Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis

Among 33 cytochrome P450s (CYPs) of Streptomyces avermitilis, CYP102D1 encoded by the sav575 gene is naturally a unique and self-sufficient CYP. Since the native cyp102D1 gene could not be expressed well in Escherichia coli, its expression was attempted using codon-optimized synthetic DNA. The gene was successfully overexpressed and the recombinant CYP102D1 was functionally active, showing a Soret peak at 450 nm in the reduced CO difference spectrum. FMN/FAD isolated from the reductase domain showed the same fluorescence in thin layer chromatography separation as the authentic standards. Characterization of the substrate specificity of CYP102D1 based on NADPH oxidation rate revealed that it catalysed the oxidation of saturated and unsaturated fatty acids with very good regioselectivity, similar to other CYP102A families depending on NADPH supply. In particular, CYP102D1 catalysed the rapid oxidation of myristoleic acid with a k(cat)/K(m) value of 453.4 ± 181.5 μM(-1)·min(-1). Homology models of CYP102D1 based on other members of the CYP102A family allowed us to alter substrate specificity to aromatic compounds such as daidzein. Interestingly, replacement of F96V/M246I in the active site increased catalytic activity for daidzein with a k(cat)/K(m) value of 100.9 ± 10.4 μM(-1)·min(-1) (15-fold).     

Export of a heterologous cytochrome P450 (CYP105D1) in Escherichia coli is associated with periplasmic accumulation of uroporphyrin

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases.     

Directed Evolution of the Actinomycete Cytochrome P450 MoxA (CYP105) for Enhanced Activity

Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did.     

Cytochrome P450(cin) (CYP176A), isolation,expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at approximately 2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450(cin), it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K(D) = 0.7 microm), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.     

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.     

Cytochrome P450 (CYP) enzymes and the development of CYP biosensors

Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the first-pass metabolism of drugs and foreign chemicals in the body. CYP reactions, therefore, are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened for CYP activity. CYP reactions in vivo require the cofactor NADPH as the source of electrons and an additional enzyme, cytochrome P450 reductase (CPR), as the electron transfer partner; consequently, any laboratory or industrial use of CYPs is limited by the need to supply NADPH and CPR. However, immobilizing CYPs on an electrode can eliminate the need for NADPH and CPR provided the enzyme can accept electrons directly from the electrode. The immobilized CYP can then act as a biosensor for the detection of CYP activity with potential substrates, albeit only if the immobilized enzyme is electroactive. The quest to create electroactive CYPs has led to many different immobilization strategies encompassing different electrode materials and surface modifications. This review focuses on different immobilization strategies that have been used to create CYP biosensors, with particular emphasis on mammalian drug-metabolizing CYPs and characterization of CYP electrodes. Traditional immobilization methods such as adsorption to thin films or encapsulation in polymers and gels remain robust strategies for creating CYP biosensors; however, the incorporation of novel materials such as gold nanoparticles or quantum dots and the use of microfabrication are proving advantageous for the creation of highly sensitive and portable CYP biosensors.     

Alkaline serine proteinases D and E of Streptomyces griseus K-1.

Two DFP-sensitive alkaline proteinases with strong esterase activity toward Ac-(Ala)3-OMe, designated as alkaline serine proteinases D and E, were purified pronase, a protease mixture from St. griseus K-1. Each was shown to be homogeneous by acrylamide disc gel electrophoresis. The molecular weights of these enzymes were estimated to be about 27,000 be gel filtration. Studies on their actions on acyl-tl-amino acid methyl or ethyl esters indicated that proteinases D and E both exhibited a broad substrate specificity and hydrolyzed the ester bonds of esters containing Trp, Tyr, Phe, Leu, and Ala. The esterase activities of both enzymes toward Ac-(Ala)3-OMe were the highest among proteinases so far isolated from various sources. Proteinases D and E also lacked cystine residues in their molecules, being entirely different from alkaline serine proteinases A, B, and C in pronase. Some differences were , however, observed between them as regards pH stability, behavior on CM-cellulose, mobility on polyacrylamide electrophoresis, and amidase activity toward Suc-(Ala)3-pNA.     

Crystal structure of CYP105A1 (P450SU-1) in complex with 1alpha,25-dihydroxyvitamin D3

Vitamin D 3 (VD 3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD 3 requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD 3 into 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1alpha,25(OH) 2D 3. The compound 1alpha,25(OH) 2D 3 is positioned 11 A from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1-VD 3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B' and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1alpha-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD 3.     

Purification and partial characterization of multiple bromoperoxidases from Streptomyces griseus

The presence of multiple bromoperoxidases in extracts of Streptomyces griseus Tü 6 was detected. The enzyme pattern varied with the age of the culture. A haem-type bromoperoxidase (BPO 2) was always present. Additionally three nonhaem-type bromoperoxidases (BPO 1a, 1b and 3) were detected and purified to homogeneity. The Mr of non-denatured BPO 1a was 70,000 +/- 10,000 and those of BPO 1b and 3 were 90,000 +/- 5000. BPO 1a and 1b were dimers with subunit Mr values of 34,000 and 43,000, respectively. BPO 3 was a trimer with a subunit Mr of 31,000. The enzymes differed in their isoelectric points, heat stability, and Km values. In immunodiffusion experiments BPO 1a and 3 showed partial identity with the nonhaem-type bromoperoxidase from Streptomyces aureofaciens. The nonhaem-type BPO 1a, 1b and 3 had neither peroxidase nor catalase activity.     

A novel xylanase from Streptomyces sp. FA1: Purification,characterization, identification,and heterologous expression

A xylanase (XynA) was purified from the culture medium of Streptomyces sp. FA1, which was previously isolated from a bamboo retting system. XynA had a molecular mass of 43 kDa, displayed maximal activity at pH 5.5, retained 41% of its maximal activity at pH 11.0, and was stable over a wide pH range (3.0 ～ 11.0). Purified XynA was subjected to peptide mass fingerprinting, which led to the cloning of the xynA gene. The xynA gene, which encodes a mature protein of 436 amino acids, was heterologously expressed in E. coli BL21(DE3). The activity in the culture medium could reach 213.5 U/mL, which was 11.2-fold higher than that produced by Streptomyces sp. FA1. BLAST searching revealed that full-length XynA shares less than 90% identity with most of its homologues, whereas amino acids 48-436 of the enzyme share 97% identity with an open reading frame encoding a putative full-length mature xylanase from Streptomyces tendae. The truncated xynA gene, xynA ^48-436 , was cloned and expressed in E. coli, however, no xylanase activity could be detected in the culture medium. Based on these results, it is suggested that XynA is a new member of glycoside hydrolases family10 with exceptional catalytic efficiency at alkaline pH.     

In vivo regulation of histidine ammonia-lyase activity from Streptomyces griseus.

The enzyme histidine ammonia-lyase (histidase) is required for growth of Streptomyces griseus on L-histidine as the sole source of nitrogen. Histidase was induced by the inclusion of histidine in the medium, regardless of the presence of other carbon and nitrogen sources. Histidase activity was increased by a shift of culture incubation temperature from 30 to 37 degrees C. Conversely, upon induction of sporulation by either phosphate starvation or nutritional downshift, histidase underwent rapid inactivation. Nutrient replenishment fully reversed histidase inactivation while simultaneously permitting reinitiation of vegetative growth. In contrast to histidase inactivation during sporulation, histidase was activated after transition of a vegetatively growing culture to stationary phase. Although neither activation nor inactivation required de novo protein synthesis, inactivation appeared to involve a heat-labile protein. The results indicate that histidase activity is regulated in vivo by a process that responds to changes in the growth phase of the organism.     

Purification and specificity of carboxypeptidase from Streptomyces griseus K-1.

A carboxypeptidase of St. griseus K-1 (CPase S) was found to possess the specificities of both mammalian pancreatic CPase A and B. Three adsorbents for affinity chromatography were prepared by coupling l-Leu, d-Leu, and d-Arg with CH-Sepharose 4B. d-Arg-CH-Sepharose and l-Leu-CH-Sepharose retained the purified CPase S but d-Leu-CH-Sepharose did not. The activities of CPase S toward CGL and BGA were eluted in the same position. CPase S migrated as a single band on polyacrylamide gel electrophoresis and the two activities were both extracted from this band on the gel.