# Patent

## Title
Method of preparing piceatannol using bacterial cytochrome P450 and composition therefor

## Abstract

Provided is a method of preparing piceatannol, and more particularly, to a method of preparing piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof, and a composition and a kit therefor.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 13/132,420 (now abandoned), having a 371(c) date of Jun. 2, 2011, which is the U.S. national stage application under 35 U.S.C. §371 of International Application No. PCT/KR2009/001859, filed Apr. 10, 2009, which claims the benefit of Korean Application No. 10-2008-0122029 filed Dec. 3, 2008, each of which is hereby incorporated by reference to the extent it is not inconsistent with the present disclosure.


REFERENCE TO A SEQUENCE LISTING
A sequence listing containing SEQ ID NOs:1-16 is submitted herewith and is specifically incorporated by reference.
TECHNICAL FIELD
The present invention relates to a method of preparing expensive piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof.
This work was supported in part by the 21C Frontier Microbial Genomics and Application Center Program of the Ministry of Education, Science & Technology of the Republic of Korea [Project No.: MG08-0306-2-0, Title: Development of humanized bacterial monooxygenase for fine chemicals using microbial cytochrome P450 enzyme genomics].
BACKGROUND ART
Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin, which is an antitoxic substance produced by a plant tissue in response to external toxicity and found in a wide variety of dietary sources including grapes, plums, and peanuts. It exhibits beneficial effects including anti-oxidant, anti-inflammatory, cardioprotective, and anti-tumor activities (Kundu and Surh, 2008; Pirola and Fröjdö, 2008; Athar, et al., 2007). Currently, numerous preclinical findings suggest resveratrol as a promising nature's arsenal for cancer prevention and treatment. As a potential anti-cancer agent, resveratrol has been shown to inhibit or retard the growth of various cancer cells in culture and implanted tumors in vivo. The biological activities of resveratrol are found to relate to its ability in modulating various targets and signaling pathway.
Piceatannol (3,5,3′,4′-tetrahydroxystilbene) is a polyphenol found in grapes and other plants. It is known as a protein kinase inhibitor that exerts immunosuppressive and antitumorigenic activities on several cell lines, and has been shown to exert various pharmacological effects on immune and cancer cells (Kim et al, 2008b and references therein). In humans, piceatannol is produced as a major metabolite of resveratrol by CYP1B1 and CYP1A2 (Potter et al., 2002; Piver et al., 2004). In addition, the metabolism of trans-resveratrol into two major metabolites, piceatannol (3,5,3′,4′-tetrahydroxystilbene) and another tetrahydroxystilbene, was catalyzed by recombinant human CYP1A1, CYP1A2 and CYP1B1 (Piver et al., 2004).




Cytochrome P450 enzymes (P450s or CYPs) constitute a large family of enzymes that are remarkably diverse oxygenation catalysts found throughout nature, from archaea to humans (available on the internet at drnelson.utmem.edu/CytochromeP450.html). Because of their catalytic diversity and broad substrate range, P450s are attractive as biocatalysts in the production of fine chemicals, including pharmaceuticals (Guengerich 2002; Urlacher et al., 2006; Yun et al., 2007; Lamb et al., 2007). In spite of the potential use of mammalian P450s in various biotechnology fields, they are not suitable as biocatalysts because of their low stability, catalytic activity, and availability.
If a metabolite, such as piceatannol, has a biological activity, direct administration of the metabolite into a living body may be beneficial. However, large quantities of the metabolite need to be produced. If pro-drugs are converted to biologically ‘active metabolites’ by human liver P450s during the drug development process (Johnson et al., 2004), large quantities of the pure metabolites are required to understand the drug's efficacy, toxic effect, and pharmacokinetics.
The pure metabolites may be difficult to synthesize. An alternative to chemical synthesis is to use P450s to generate the metabolites of drugs or drug candidates. Hepatic microsomes can be a source of human P450s, but their limited availability make their use in preparative-scale metabolite synthesis impractical. Some human enzymes can also be obtained by expression of recombinant hosts. Metabolite preparation has been demonstrated using human P450s expressed in Escherichia coli and in insect cells (Parikh et al., 1997; Rushmore et al., 2000; Vail et al., 2005), but these systems are costly and have low productivities due to limited stabilities and slow reaction rates (usually <5 min−1 (Guengerich et al., 1996)). An alternative approach to preparing the human metabolites is to use an engineered bacterial P450 that has the appropriate specificity.
The P450 BM3 (CYP102A1) from Bacillus megaterium has strong similarity to eukaryotic members of the CYP4A (fatty acid hydroxylase) family. It was shown that engineered CYP102A1 mutants could oxidize several human P450 substrates to produce the authentic metabolites with higher activities (Kim et al., 2008; Otey et al., 2005; Yun et al., 2007 and references therein). Furthermore, CYP102A1 is a versatile monooxygenase with a demonstrated ability to work on a diversity of substrates (Bernhardt et al., 2006, Di Nardo et al., 2007).
Recently, wild-type CYP102A1 has been engineered to oxidize compounds showing little or no structural similarity to its natural substrate fatty acids (Lamb et al., 2007). The compounds include testosterone, several drug-like molecules, and polycyclic aromatic hydrocarbons (PAHs), which are known substrates of human P450 enzymes (Carmichael et al., 2001; van Vugt-Lussenburg et al., 2006). However, there has been no research on whether resveratrol can be used as a substrate. A set of CYP102A1 mutants was shown to generate larger quantities of the authentic human metabolites of drugs, which may be difficult to synthesize (Otey et al., 2005). An alternative approach to preparing the metabolites is to use engineered CYP102A1 enzymes with desired properties.
Based on the scientific literature, several amino acid residues in CYP102A1 were mutated to generate mutant enzymes showing increased activity toward human P450 substrates (Yun et al., 2007). Very recently, it was reported that some selected mutations enabled the CYP102A1 enzyme to catalyze O-deethylation and 3-hydroxylation of 7-ethoxycoumarin, which are the same reactions catalyzed by human P450s (Kim et al., 2008a).
There are several patent applications related to piceatannol. That is, a composition for antihypertensive effects comprising a Rhei Rhizome extract or active compounds isolated therefrom is disclosed in Korean Patent Application No. 10-2005-0126879, and a cosmetic composition containing piceatannol and vitamin A is disclosed in Korean Patent Application No. 10-2007-0025087. However, a patent application related to a method of preparing piceatannol has not been filed. While a method of chemically synthesizing resveratrol and piceatannol is disclosed in WO2008012321, a method of biologically preparing resveratrol and piceatannol has not been disclosed.
All cited references are incorporated herein by reference in their entireties. The information disclosed herein is intended to assist the understanding of technical backgrounds of the present invention, and cannot be prior art.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
The present invention provides a method of preparing piceatannol using an enzyme which stably and efficiently catalyzes oxidation of resveratrol into piceatannol.
Technical Solution
While searching for a method of preparing piceatannol, the present inventors found that bacterial P450 (CYP102A1) and mutants thereof may be selectively used to produce metabolites of resveratrol in humans, particularly piceatannol.
Advantageous Effects
The present invention provides a method of producing large quantities of piceatannol, which is about 60 times more expensive than resveratrol, from resveratrol, and a composition and a kit therefor.
While metabolites of resveratrol produced using the human CYP1A2 include piceatannol and other hydroxylated products, piceatannol may be selectively produced by CYP102A1 or mutants thereof.
In addition, in an in vitro system, human CYP1A2 may be inactivated by the metabolites of the human CYP1A2 itself. However, wild-type CYP102A1 or mutants of CYP102A1 may not be inactivated by the metabolites.
Even though chemical synthesis of piceatannol has been reported, biological synthesis of piceatannol using enzymes is effective and environmentally friendly in terms of white biotechnology.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates HPLC chromatograms of resveratrol metabolites produced by human CYP1A2 and bacterial CYP102A1 mutants (A: resveratrol and piceatannol standards, B: human CYP1A2, C: Mutant #10, D: Mutant #13, E: Mutant #14, and F: Mutant #15). Peaks of the substrate resveratrol and two major products are indicated. UV absorbance was monitored at 320 nm.
FIG. 2 illustrates GC analysis results of resveratrol metabolite derivatives produced by CYP102A1 and mutants thereof (A: trans-resveratrol and piceatannol standards, B: human CYP1A2, C: Mutant #10, D: Mutant #13, E: Mutant #14, and F: Mutant #15). The mass spectra of the reaction samples showed peaks at 28.09 min (resveratrol) and 32.98 min (piceatannol).
FIG. 3 illustrates GC elution profiles (A) and MS spectra (B: resveratrol, C and D: resveratrol metabolites) of resveratrol metabolite derivatives produced by human CYP1A2.
FIG. 4 illustrates MS spectra of peaks of metabolites produced by standard trans-resveratrol (A) and piceatannol (B) that were eluted at 29.08 min and 32.98 min (Res-TMS; m/z=444, Pic-TMS; m/z=532), peaks of metabolites produced by human CYP1A2 that were eluted at 29.08 min and 32.98 min (C: Res-TMS; m/z=444, D: Pic-TMS; m/z=532), and peaks of metabolites produced by CYP102A1 mutants that were eluted at 32.98 min (Pic-TMS; m/z=532) (E: Mutant #10, F: Mutant #13, G: Mutant #14, and H: Mutant #15).
FIG. 5 illustrates total turnover numbers (TTN; mol product/mol catalyst) of piceatannol formation by CYP102A1 mutants. 100 μM trans-resveratrol was used. The reaction was initiated by the addition of the NADPH-generating system, incubated for 1 or 2 hours, respectively, at 30° C. The formation rate of piceatannol was determined by HPLC.
FIG. 6 illustrates the stability of P450 enzymes measured by CO-difference spectra during the oxidation of resveratrol by the P450 enzymes in the presence of NADPH. Value of 100% represents the P450 concentration before the incubation of the reaction mixture. Mutants #10, 11, 14, and 15 showed the highest stability.
FIG. 7 shows amino acid sequence of CYP102A1 (SEQ ID NO:16).



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention provides a method of preparing piceatannol, and more particularly, a method of preparing piceatannol from resveratrol using bacterial cytochrome P450 BM3 (CYP102A1) or mutants thereof, and a composition and a kit therefor.
The present inventors have found that bacterial CYP102A1 and mutants thereof may act as a catalyst for oxidation of resveratrol known as a substrate of human P450. In particular, while metabolites of resveratrol produced by the human CYP1A2, as a catalyst, include piceatannol and other hydroxylated products, piceatannol may be selectively produced by CYP102A1 or mutants thereof, as a catalyst. Although human CYP1A2 is inactivated in vitro by the metabolites, wild-type CYP102A1 or mutants of CYP102A1 are not inactivated in vitro by the metabolites.
Specifically, the present inventors have identified that trans-resveratrol is converted into hydroxylated metabolites when large quantities of wild-type CYP102A1 and site-directed mutants of CYP102A1 are expressed in E. coli (Tables 1 and 2), and the wild-type and mutants of CYP102A1 are subjected to a reaction with trans-resveratrol and an NADPH-generating system, using HPLC (FIG. 1) and GC-MS spectra (FIGS. 2 to 5). The human CYP1A2 oxidizes resveratrol to produce two major metabolites: piceatannol and other hydroxylated products. On the other hand, wild-type CYP102A1 and mutants of CYP102A1 selectively produce only one major metabolite: piceatannol.
Turnover numbers of trans-resveratrol oxidation (piceatannol formation) by CYP102A1 mutants are measured. As a result, mutants #8 to #17 showed higher catalytic activities than that of wild-type CYP102A1. Mutant #13 has about 18-fold higher activity than the wild-type CYP102A1 (Table 3). After 1 or 2 hours of hydroxylation of resveratrol by CYP102A1 mutants, the total turnover number (TTN; mol product/mol catalyst) of the piceatannol formation is measured. As a result, mutant #13 shows the highest activity and has about 10-fold higher activity than human CYP1A1. The amount of products obtained by 1 hour-hydroxylation by human CYP1A2 is less than that obtained by 2 hour-hydroxylation by human CYP1A2. This is inferred because the human CYP1A2 is unstable or the activity of the human CYP1A2 is inhibited by the metabolites.
Kinetic parameters for 3′-hydroxylation of resveratrol by wild-type CYP102A1 and mutants of CYP102A1 are measured. Mutant #13 shows the highest kcat and Km and the highest catalytic efficiency (kcat/Km) (Table 4). In case of human CYP1A2, kinetic parameters could not be obtained. This is inferred because human P450 is inactivated by the metabolites of human P450 itself. Thus, human CYP1A2 may not be used in an in vitro system, but wild-type CYP102A1 or mutants of CYP102A1 may be used therein.
Resveratrol acts as a substrate and an inhibitor to human CYP1A2 at the same time. Piceatannol, the major metabolite of resveratrol, is also known as an inhibitor to human CYP1A2. Thus, the stability of P450 enzymes is measured during oxidation of resveratrol by P450 enzymes in the presence of NADPH using CO-difference spectra. As a result, mutants #10, 11, 14, and 15 show the highest stability (FIG. 6).
Based on these results, the present invention provides a composition for preparing piceatannol from resveratrol, the composition including wild-type CYP102A1 and/or mutants of CYP102A1.
The present invention also provides a method of preparing piceatannol, the method including reacting at least one enzyme selected from a group consisting of wild-type and mutants of CYP102A1 with resveratrol. The method may further include adding an NADPH-generating system.
The present invention also provides a kit for preparing piceatannol from resveratrol, the kit including at least one enzyme selected from a group consisting of wild-type CYP102A1 and mutants of CYP10212 and an NADPH-generating system. The kit may further include a reagent required for the progression of the reaction.
The NADPH-generating system used for the method and the kit may be any known system. For example, the NADPH-generating system may be glucose 6-phosphate dehydrogenase, NADP+, and yeast glucose 6-phosphate, but is not limited thereto.
The piceatannol formation may be performed at a temperature ranging from about 0° C. to about 40° C., preferably from about 30° C. to about 40° C.
Mutagenesis of CYP102A1 may be performed using known methods such as deletion mutagenesis (Kowalski D. et al., J. Biochem., 15, 4457), PCT method, Kunkel method, site-directed mutagenesis, DNA shuffling, StEP (staggered extension process), error-prone PCR, etc.
The mutants of CYP102A1 may have a sequence modified by natural or artificial substitution, deletion, addition, and/or insertion of amino acid of the wild-type CYP102A1. The amino acid may be substituted with an amino acid with similar properties. For example, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan are non-polar amino acids with similar properties. Neutral amino acids are glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, acidic amino acids are aspartic acid, glutamic acid, and basic amino acids are lysine, arginine, and histidine.
The mutants of CYP102A1 include polypeptide with an amino acid sequence which is more than 50% similar, preferably more than 75% similar, and more preferably more than 90% similar to the sequence of wild-type CYP102A1.
The mutants of CYP102A1 may be prepared by at least one selected from a group consisting of: substituting 47th amino acid arginine (R) of wild-type CYP102A1 with one amino acid selected from a group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan, substituting 51st amino acid tyrosine (Y) of wild-type CYP102A1 with one amino acid selected from a group consisting of phenylalanine, alanine, valine, leucine, isoleucine, proline, methionine, tryptophan, substituting 64th amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acid selected from a group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, substituting 74th amino acid alanine (A) of wild-type CYP102A1 with one amino acid selected from a group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, substituting 81st amino acid phenylalanine (F) of wild-type CYP102A1 with one amino acid selected from a group consisting of alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan, substituting 86th amino acid leucine (L) of wild-type CYP102A1 with one amino acid selected from a group consisting of alanine, valine, isoleucine, proline, methionine, phenylalanine, and tryptophan, substituting 87th amino acid phenylalanine (F) of wild-type CYP102A1 with one amino acid selected from a group consisting of alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan, substituting 143rd amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acid selected from a group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, substituting 188th amino acid leucine (L) of wild-type CYP102A1 with one amino acid selected from a group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and substituting 267th amino acid glutamic acid (E) of wild-type CYP102A1 with one amino acid selected from a group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.
Preferably, the mutants of CYP102A1 may be prepared by at least one selected from a group consisting of: substituting 47th amino acid arginine (R) of wild-type CYP102A1 with leucine (L), substituting 51st amino acid tyrosine (Y) of wild-type CYP102A1 with phenylalanine, substituting 64th amino acid glutamic acid (E) of wild-type CYP102A1 with glycine (G), substituting 74th amino acid alanine (A) of wild-type CYP102A1 with glycine (G), substituting 81st amino acid phenylalanine (F) of wild-type CYP102A1 with isoleucine (I), substituting 86th amino acid leucine (L) of wild-type CYP102A1 with isoleucine (I), substituting 87th amino acid phenylalanine (F) of wild-type CYP102A1 with valine (V), substituting 143rd amino acid glutamic acid (E) of wild-type CYP102A1 with glycine (G), substituting 188th amino acid leucine (L) of wild-type CYP102A1 with glutamine (Q), and substituting 267th amino acid glutamic acid (E) of wild-type CYP102A1 with valine (V).
More preferably, the mutants of CYP102A1 may include amino acid substitution sites of wild-type CYP102A1 selected from a group consisting of F87A, R47L/Y51F, A74G/F87V/L188Q, R47L/L86I/L188Q, R47L/F87V/L188Q, R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V, R47L/L86I/F87V/L188Q, R47L/F87V/E143G/L188Q/E267V, R47L/E64G/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/L188Q/E267V, and R47L/E64G/F81I/F87V/E143G/L188Q/E267V.
Protein according to the present invention may be prepared using methods known in the art, for example, genetic engineering techniques, solid-phase peptide synthesis (Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)), or method of cleaving protein using peptidases. Protein according to the present invention may be natural protein, or may be prepared by a recombination of culturing cells transformed with DNA encoding CYP102A1 or mutants thereof and collecting the protein. Protein may be prepared by inserting nucleic acid molecules encoding protein according to the present invention into an expression vector, transforming the vector into a host cell, and purifying protein expressed by the transformed host cell.
The vector may be plasmid, cosmid, a virus, or phage. The host cell into which DNA in the vector is cloned or expressed may be a prokaryotic cell, a yeast cell, and a eukaryotic cell. Culture conditions such as a culture medium, temperature, and pH may be selected by those of ordinary skill in the art without undue experiment. In general, principles, protocols, and techniques to maximize productivity of the culture of cells are disclosed in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991).
The expression and cloning vector may include a promoter operationally linked to a nucleic acid sequence that encodes CYP102A2 or mutants thereof which induce the synthesis of mRNA. Various promoters recognized by host cells are known. A promoter suitable for a prokaryotic host cell may be β-lactamase and a lactose promoter system, alkali phosphatase, a tryptophan (trp) promoter system, and a hybrid promoter, for example a tac promoter. In addition, the promoter used in bacterial systems may include a Shine-Dalgarno (SD) sequence operationally linked to DNA that encodes protein. A promoter suitable for a yeast host cell may include 3-phosphoglycerate kinase or other glucosidases.
The present invention will now be described in greater detail with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLES
Trans-resveratrol, trans-piceatannol, and NADPH were purchased from Sigma-Aldrich (Milwaukee, Wis., USA). Other chemicals were of the highest grade commercially available. Human CYP1A2 was prepared as disclosed by Kim et al., (2008c).
Example 1
Construction of P450 BM3 Mutants by Site-Directed Mutagenesis
Site-directed mutants of CYP102A1 were prepared as disclosed by Kim et al., Drug Metab Dispos, volume 35, p. 2166-2170, 2008. PCR primers used to introduce BamHI/SacI restriction sites and to induce mutation are listed in Table 1. Codons for amino acid substitution are in italics and underlined. The PCR primers were obtained from Genotech (Daejeon, Korea). Genes that encode CYP102A1 mutants were amplified from pCWBM3 by PCR using primers designed to facilitate cloning into an expression vector pCWori (Dr. F. W. Dahlquist, University of California, Santa Barbara, Calif.) or pSE420 (Invitrogen) (Farinas et al., 2001). Oligonucleotide assembly was performed by PCR using the 14 sets of designed primers listed in Table 1. The amplified genes were subsequently cloned into the PCWBM3 BamHI/SacI vector at the BamHI/SacI restriction sites. These plasmids were transformed into Escherichia coli DH5α F′IQ (Invitrogen), and this strain was also used to express the mutant CYP102A1 proteins. After mutagenesis, the presence of the desired mutations was confirmed by DNA sequencing in Genotech (Daejeon, Korea).







TABLE 1







Primers used for the generation of mutants


in this study








Name
Sequence





BamHI
5′-AGC GGA TCC ATG ACA ATT AAA GAA 


forward
ATG CCT C-3′


 


SacI
5′-ATC GAG CTC GTA GTT TGT AT-3′


reverse



 


R47L
5′-GCG CCT GGT CTG GTA ACG CG-3′


 


Y51F
5′-GTA ACG CGC TTC TTA TCA AGT-3′


 


E64G
5′-GCA TGC GAT GGC TCA CGC TTT-3′


 


A74G
5′-TAAGT CAA GGC CTT AAA TTT GTA CG-3′


 


F81I
5′-GTA CGT GAT ATT GCA GGA GAC-3′


 


L86I
5′-GGA GAC GGG ATT TTT ACA AGC T-3′


 


F87A
5′-GAC GGG TTA GCG ACA AGC TGG-3′


 


F87V
5′-GAC GGG TTA GTG ACA AGC TGG-3′


 


E143G
5′-GAA GTA CCG GGC GAC ATG ACA-3′


 


L188Q
5′-ATG AAC AAG CAG CAG CGA GCA A-3′


 


A264G
5′-TTC TTA ATT GGG GGA CAC GTG-3′


 


E267V
5′-T GCG GGA CAC GTG ACA ACA AGT-3′


 


L86I/F87V
5′-GGA GAC GGG ATT GTG ACA AGC TG-3′









Example 2
Expression and Purification of Wild-Type CYP102A1 and Mutants of CYP102A1
Plasmids comprising a gene of wild-type CYP102A1 and mutants of CYP102A1 (pCWBM3) were transformed into Escherichia coli DH5α F′-IQ. A single colony was inoculated into 5 ml of a Luria-Bertani medium supplemented with ampicillin (100 g/ml) and cultured at 37° C. This culture was inoculated into 250 ml of a Terrific Broth medium supplemented with ampicillin (100 g/ml). The cells were grown at 37° C. while shaking at 250 rpm to an OD600 of up to 0.8, at which gene expression was induced by the addition of isopropyl-δ-D-thiogalactopyranoside to a final concentration of 0.5 mM. δ-Aminolevulinic acid (1.0 mM) was also added thereto. Following induction, the cultures were allowed to grow another 36 hours at 30° C. Cells were harvested by centrifugation (15 min, 5000 g, 4° C.). The cell pellet was resuspended in a TES buffer (100 mM Tris-HCl, pH 7.6, 500 mM sucrose, 0.5 mM EDTA) and lysed by sonication (Sonicator; Misonix, Inc., Farmingdale, N.Y.). After the lysate was centrifuged at 100,000 g (90 min, 4° C.), a soluble cytosolic fraction was collected and used for the activity assay. The soluble cytosolic fraction was dialyzed against a 50 mM potassium phosphate buffer (pH 7.4) and stored at −80° C. Enzymes were used within 1 month of manufacture.
CYP102A1 concentrations were determined from the CO-difference spectra as described by Omura and Sato (1964) using ε=91 mM/cm. For all of the wild-type enzymes and mutated enzymes, a typical culture yielding of 300 to 700 nM P450 enzymes could be detected. The expression level of wild-type CYP102A1 and mutants of CYP102A1 was in the range of 1.0 to 2.0 nmol P450/mg cytosolic protein.
Several mutants with high catalytic activity for humans were selected, and the substitution sites in the mutants are shown in Table 2 below.







TABLE 2







CYP102A1 mutants used in this study









Abbreviations
BM3 wild type and mutants
Ref.





WT
BM3 wild type



Mutant #1
F87A
Carmichael et al., 2001


Mutant #2
A264G
Carmichael et al., 2001


Mutant #3
F87A/A264G
Carmichael et al., 2001


Mutant #4
R47L/Y51F
Carmichael et al., 2001


Mutant #5
R47L/Y51F/A264G
Carmichael et al., 2001


Mutant #6
R47L/Y51F/F87A
Carmichael et al., 2001


Mutant #7
R47L/Y51F/F87A/A264G
Carmichael et al., 2001


Mutant #8
A74G/F87V/L1188Q
Li et al., 2001


Mutant #9
R47L/L86I/L188Q
Kim et al., 2008a


Mutant #10
R47L/F87V/L188Q
van Vugt-Lussenburg




et al., 2007


Mutant #11
R47L/F87V/L188Q/E267V
van Vugt-Lussenburg




et al., 2007


Mutant #12
R47L/L86I/L188Q/E267V
Kim et al., 2008


Mutant #13
R47L/L86I/F87V/L188Q
van Vugt-Lussenburg




et al., 2007


Mutant #14
R47L/F87V/E143G/L188Q/
Kim et al., 2008a



E267V


Mutant #15
RA7L/E64G/F87V/E143G/
Kim et al., 2008a



L188Q/E267V


Mutant #16
R47L/F81I/F87V/E143G/
Kim et al., 2008a



L188Q/E267V


Mutant #17
R47L/E64G/F81I/F87V/
van Vugt-Lussenburg



E143G/L188Q/E267V
et al., 2007









Example 3
Hydroxylation of Trans-Resveratrol by Wild-Type P450 BM3 and Mutants of P450 BM3
Oxidation of trans-resveratrol, a substrate of human CYP1A2, by CYP102A1 was identified. Typical steady-state reactions for trans-resveratrol hydroxylation included 50 pmol P450 BM3 in 0.25 ml of a 100 mM potassium phosphate buffer (pH 7.4) were performed along with a specified amount of a substrate. To determine the kinetic parameter of several CYP102A1 mutants, 2 to 100 μM of trans-resveratrol was used. An NADPH-generating system was used to initiate reaction solutions (final concentrations: 10 mM glucose 6-phosphate, 0.5 mM NADP+, and 1 IU yeast glucose 6-phosphate per ml). Trans-resveratrol stocks (20 mM) were prepared in DMSO and diluted into the enzyme reactions with the final organic solvent concentration <1% (v/v). Reactions were generally incubated for 10 min at 37° C., and terminated with 105 μl of ice-cold acetic acid/methanol (95/5, v/v).
Example 3-1
HPLC Analysis
After centrifugation of the reaction mixture, the supernatant was analyzed by HPLC (Piver et al. 2004). Samples (30 μl) were injected into a Gemini C18 column (4.6 mm×150 mm, 5 μm, Phenomenex, Torrance, Calif.). The mobile phase A was water containing 87 mM of 0.5% acetic acid/acetonitrile (95/5, v/v); whereas the mobile phase B was acetonitrile/0.5% acetic acid (95/5, v/v). The mobile phase A/B (75/25, v/v) was delivered at a flow rate of 1 ml·min−1 by a gradient pump (LC-20AD, Shimadzu, Kyoto, Japan). Eluates were detected by UV rays at 320 nm.
FIG. 1 illustrates HPLC chromatograms of resveratrol metabolites produced by human CYP1A2 and bacterial CYP102A1 mutants (A: resveratrol and piceatannol standards, B: human CYP1A2, C: Mutant #10, D: Mutant #13, E: Mutant #14, and F: Mutant #15). Peaks of the substrate resveratrol and two major products are indicated. UV absorbance was monitored at 320 nm.
While human CYP1A2 oxidized resveratrol to produce two major metabolites: piceatannol and other hydroxylated products (B), wild-type CYP102A1 and mutants of CYP102A1 produced only one major metabolite. The retention time of the peak was exactly matched to that of the piceatannol standard. That is, the wild-type CYP102A1 and mutants of CYP102A1 selectively produced piceatannol when oxidizing resveratrol. Since there is no need to separate the hydroxylated product from piceatannol, the use of wild-type CYP102A1 and mutants of CYP102A1 is beneficial.
Example 3-2
GC-MS Analysis
For the identification of resveratrol metabolite, produced by P450 BM3 mutants, GC-MS analysis was done by comparing GC-profiles and fragmentation patterns of piceatannol and resveratrol. An oxidation reaction of trans-resveratrol by P450 BM3 mutants was done. The aqueous samples were extracted with ethyl acetate. After centrifugation, the organic phase was dried under nitrogen as well as the standard trans-resveratrol and piceatannol solutions (10 mM in DMSO). Then, trimethylsilyl (TMS) derivatives were prepared as follows. 100 μl of a solution of BSTFA/TMCS (99/1, v/v) (Supelco) was added to the dry residue or standard trans-resveratrol and piceatannol, and then the mixture was left for 60 min at 60° C.
GC-MS analysis was performed on a GC-2010 gas chromatograph (Shimadzu, Kyoto, Japan) with an Rtx-5 (5% diphenyl/95% dimethyl polysiloxane capillary column) (30 m×0.32 mm i.d.×0.25 μm film thickness). The injector temperature was 250° C. The derivatives of resveratrol and piceatannol were separated by GC analysis under the conditions: GC oven conditions of 60° C. for 5 min, followed by an increase of 50° C.·min−1 up to 200° C. and then 2° C.·min−1 up to 300° C. The gas chromatography was combined with a GCMS-QP2010 Shimazu mass spectrometer operating in an electron ionization mode (70 eV) (Piver et al. 2004).
FIG. 2 illustrates GC analysis results of resveratrol metabolite derivatives produced by CYP102A1 and mutants thereof (A: standard trans-resveratrol and piceatannol, B: human CYP1A2, C: Mutant #10, D: Mutant #13, E: Mutant #14, and F: Mutant #15). The mass spectra of the reaction samples showed peaks at 28.09 min (resveratrol) and 32.98 min (piceatannol).
As a result of the GC-MS analysis, it was identified that the retention time and fragmentation patterns of the metabolite produced by the CYP102A1 mutants were exactly matched to those of the piceatannol standard, and thus the metabolite produced by the CYP102A1 mutants was piceatannol. Although piceatannol and other hydroxylated products were found as the two major metabolites of resveratrol by human liver microsomes, all wild-type CYP102A1 and mutants of CYP102A1 showed only one hydroxylated product, i.e., piceatannol. The human P450 1A2, the major enzyme for hydroxylation reactions of resveratrol in human liver, also showed a preference for the 3′-hydroxylation reaction over the hydroxylation reaction at other position humans (B of FIG. 2 and FIG. 4). However, unlike the human P450 enzyme, wild-type CYP102A1 and mutants of CYP102A1 produced only piceatannol without producing other hydroxylated products.
FIG. 3 illustrates GC elution profiles (A) and MS spectra (B: resveratrol, C and D: resveratrol metabolites) of trans-resveratrol metabolite derivatives produced by human CYP1A2.
FIG. 4 illustrates MS spectra of peaks of metabolites produced by standard trans-resveratrol (A) and piceatannol (B) that were eluted at 29.08 min and 32.98 min (Res-TMS; m/z=444, Pic-TMS; m/z=532), peaks of metabolites produced by human CYP1A2 that were eluted at 29.08 min and 32.98 min (C: Res-TMS; m/z=444, D: Pic-TMS; m/z=532), and peaks of metabolites produced by CYP102A1 mutants that were eluted at 32.98 min (Pic-TMS; m/z=532) (E: Mutant #10, F: Mutant #13, G: Mutant #14, and H: Mutant #15).
Example 3-3
Determination of Total Turnover Number
To determine the total turnover number of CYP102A1 mutants, 100 μM of trans-resveratrol was used. The reaction was initiated by the addition of the NADPH-generating system, incubated for 1 and 2 hours, respectively, at 30° C. The formation rate of piceatannol was determined by HPLC as described above.
Table 3 shows turnover numbers of 17 mutants for trans-resveratrol oxidation (piceatannol formation). The ability of wild-type P450 BM3 and mutants of P450 BM3 to oxidize trans-resveratrol was measured at a fixed substance concentration (100 μM).







TABLE 3







Rates of piceatannol formation by various CYP102A1 mutantsa











nmol product/min/nmol P450



Enzyme
Piceatannol







WT
0.22 ± 0.01



Mutant



#1
0.021 ± 0.001



#2
NDb



#3
NDb



#4
0.027 ± 0.001



#5
NDb



#6
NDb



#7
NDb



#8
1.1 ± 0.1



#9
0.24 ± 0.01



#10 
1.6 ± 0.1



#11 
0.64 ± 0.03



#12 
0.33 ± 0.03



#13 
4.0 ± 0.1



#14 
1.3 ± 0.1



#15 
1.9 ± 0.3



#16 
0.97 ± 0.1 



#17 
1.8 ± 0.1










Mutants #8 to #17 showed higher catalytic activities than that of wild-type CYP102A1. Mutant #13 showed about 18-fold higher activity than the wild-type CYP102A1.
FIG. 5 illustrates total turnover numbers (TTN; mol product/mol catalyst) piceatannol formation by CYP102A1 mutants. 100 μM trans-resveratrol was used. The reaction was initiated by the addition of the NADPH-generating system, incubated for 1 or 2 hours, respectively, at 30° C. The formation rate of piceatannol was determined by HPLC as described above.
Mutant #13 showed the highest activity and has about 10-fold higher activity than human CYP1A1. Meanwhile, the amount of products obtained by 1 hour-hydroxylation by human CYP1A2 is less than that obtained by 2 hour-hydroxylation by human CYP1A2. This was inferred because CYP1A2 is unstable or the activity of the human CYP1A2 is inhibited by the metabolites.
Example 3-4
Determination of Kinetic Parameters
Kinetic parameters (Km and kcat) were determined using nonlinear regression analysis with GraphPad PRISM software (GraphPad, San Diego, Calif., USA). The data were analyzed using the standard Michaelis-Menten equation: v=kcat[E][S]/([S]+Km), where the velocity of the reaction is a function of the rate-limiting step in turnover (kcat), the enzyme concentration ([E]), substrate concentration ([S]), and the Michaelis constant (Km).
Table 4 shows kinetic parameters for 3′-hydroxylation of resveratrol by wild-type CYP102A1 and mutants of CYP102A1.







TABLE 4







Kinetic parameters of piceatannol


formation by CYP102A1 mutants









Piceatannol formation












P450 BM3
kcat(min−1)
Km(μM)
kcat/Km







Mutant #9
0.20 ± 0.02
66 ± 14
0.0030 ± 0.0007



Mutant #10
1.1 ± 0.1
30 ± 13
0.037 ± 0.016



Mutant #11
0.12 ± 0.01
2.7 ± 0.7
0.044 ± 0.012



Mutant #12
0.13 ± 0.01
54 ± 11
0.0024 ± 0.0005



Mutant #13
6.7 ± 0.3
15 ± 3 
0.45 ± 0.09



Mutant #14
0.58 ± 0.04
13 ± 3 
0.046 ± 0.011



Mutant #15
0.54 ± 0.02
16 ± 2 
0.038 ± 0.005










Mutant #13 showed the highest kcat and Km and the highest catalytic efficiency (kcat/Km).
In case of human CYP1A2, kinetic parameters could not be obtained. This is inferred because P450 is inactivated by the metabolites of P450 itself (Chun et al., 1999, 2001). Thus, human CYP1A2 may not be used in an in vitro system, but wild-type CYP102A1 or mutants of CYP102A1 may be used therein.
Example 4
Stability of CYP102A1 Mutants
It is known that metabolites of resveratrol are potent inhibitors against human CYPs, i.e., CYP1A1, 1A2, and 1B1 (Chun et al., 1999, 2001). Resveratrol acts as a substrate and inhibitor to human CYP1A2 at the same time (Fairman et al., 2007; Piver et al., 2004). Piceatannol, the major metabolite of resveratrol, is also known as a potent inhibitor to human CYP1A2 (Mikstacka et al., 2006).
FIG. 6 illustrates the stability of P450 enzymes measured by CO-difference spectra during the oxidation of resveratrol by the P450 enzymes in the presence of NADPH. Value of 100% represents the P450 concentration before the incubation of the reaction mixture. Mutants #10, 11, 14, and 15 showed the highest stability.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
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## Claims

1. A method for preparing piceatannol, the method comprising reacting at least one mutant of CYP102A1 with resveratrol, wherein CYP102A1 is a cytochrome P450 from B. megaterium, wherein the mutant of CYP102A1 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 16, wherein the amino acid sequence of the mutant of CYP102A1 differs from SEQ ID NO: 16 by one or more amino acid substitutions at positions 47, 51, 64, 74, 81, 86, 87, 143, 188 and/or 267 of SEQ ID NO: 16, wherein the mutant of CYP102A1 catalyzes the conversion of resveratrol to piceatannol, and wherein the one or more substitutions are selected from the group consisting of:
substitution of the amino acid corresponding to arginine at position 47 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan,
substitution of the amino acid corresponding to tyrosine at position 51 of SEQ ID NO: 16 with one amino acid selected from the group consisting of phenylalanine, alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan,
substitution of the amino acid corresponding to glutamic acid at position 64 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine,
substitution of the amino acid corresponding to alanine at position 74 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine,
substitution of the amino acid corresponding to phenylalanine at position 81 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan,
substitution of the amino acid corresponding to leucine at position 86 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, isoleucine, proline, methionine, phenylalanine, and tryptophan,
substitution of the amino acid corresponding to phenylalanine at position 87 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan,
substitution of the amino acid corresponding to glutamic acid at position 143 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine,
substitution of the amino acid corresponding to leucine at position 188 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and
substitution of the amino acid corresponding to glutamic acid at position 267 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.

2. The method of claim 1, further comprising adding an NADPH-generating system.

3. The method of claim 2, wherein the NADPH-generating system comprises glucose 6-phosphate dehydrogenase, NADP+, and yeast glucose 6-phosphate.

4. The method of claim 1, wherein the resveratrol is trans-resveratrol.

5. The method of claim 1, wherein the one or more substitutions are selected from the group consisting of:
substitution of the amino acid corresponding to arginine at position 47 of SEQ ID NO: 16 with leucine (L),
substitution of the amino acid corresponding to tyrosine at position 51 of SEQ ID NO: 16 with phenylalanine (F),
substitution of the amino acid corresponding to glutamic acid at position 64 of SEQ ID NO: 16 with glycine (G),
substitution of the amino acid corresponding to alanine at position 74 of SEQ ID NO: 16 with glycine (G),
substitution of the amino acid corresponding to phenylalanine at position 81 of SEQ ID NO: 16 with isoleucine (I),
substitution of the amino acid corresponding to leucine at position 86 of SEQ ID NO: 16 with isoleucine (I),
substitution of the amino acid corresponding to phenylalanine at position 87 of SEQ ID NO: 16 with valine (V),
substitution of the amino acid corresponding to glutamic acid at position 143 of SEQ ID NO: 16 with glycine (G),
substitution of the amino acid corresponding to leucine at position 188 of SEQ ID NO: 16 with glutamine (Q), and
substitution of the amino acid corresponding to glutamic acid at position 267 of SEQ ID NO: 16 with valine (V).

6. The method of claim 1, wherein the mutant of CYP102A1 comprises amino acid substitutions that correspond to amino acid substitutions in the CYP102A1 of SEQ ID NO: 16, wherein said substitutions are selected from the group consisting of F87A, R47L/Y51F, A74G/F87V/L188Q, R47L/L86I/L188Q, R47L/F87V/L188Q, R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V, R47L/L86I/F87V/L188Q, R47L/F87V/E143G/L188Q/E267V, R47L/E64G/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/L188Q/E267V, and R47L/E64G/F81I/F87V/E143G/L188Q/E267V.

7. The method of claim 1, wherein the reacting step selectively produces piceatannol without producing other hydroxylated products.

8. The method of claim 1, wherein the method is carried out in an in vitro system and metabolites produced by the reaction do not inactivate the mutants of CYP102A1.

9. A method for making piceatannol from resveratrol, the method comprising the steps of:
providing a catalyst comprising a mutant of CYP102A1, wherein CYP102A1 is a cytochrome P450 from B. megaterium; and
reacting resveratrol in the presence of the catalyst and an NADPH-generating system; wherein the reacting step selectively produces one major metabolite that is piceatannol,
wherein the mutant of CYP102A1 comprises an amino acid sequence at least 95% identical to SEQ ID NO: 16, wherein the amino acid sequence of the mutant of CYP102A1 differs from SEQ ID NO: 16 by one or more amino acid substitutions at positions 47, 51, 64, 74, 81, 86, 87, 143, 188 and/or 267 of SEQ ID NO: 16, wherein the mutant of CYP102A1 catalyzes the conversion of resveratrol to piceatannol, and wherein the one or more substitutions are selected from the group consisting of:
substitution of the amino acid corresponding to arginine at position 47 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan,
substitution of the amino acid corresponding to tyrosine at position 51 of SEQ ID NO: 16 with one amino acid selected from the group consisting of phenylalanine, alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan,
substitution of the amino acid corresponding to glutamic acid at position 64 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine,
substitution of the amino acid corresponding to alanine at position 74 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine,
substitution of the amino acid corresponding to phenylalanine at position 81 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan,
substitution of the amino acid corresponding to leucine at position 86 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, isoleucine, proline, methionine, phenylalanine, and tryptophan,
substitution of the amino acid corresponding to phenylalanine at position 87 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, and tryptophan,
substitution of the amino acid corresponding to glutamic acid at position 143 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine,
substitution of the amino acid corresponding to leucine at position 188 of SEQ ID NO: 16 with one amino acid selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and
substitution of the amino acid corresponding to glutamic acid at position 267 of SEQ ID NO: 16 with one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.

10. The method of claim 9, wherein the resveratrol is trans-resveratrol.

11. The method of claim 9, wherein the CYP102A1 comprises SEQ ID NO: 16.

12. The method of claim 9, wherein the one or more substitutions are selected from the group consisting of:
substitution of the amino acid corresponding to arginine at position 47 of SEQ ID NO: 16 with leucine (L),
substitution of the amino acid corresponding to tyrosine at position 51 of SEQ ID NO: 16 with phenylalanine (F),
substitution of the amino acid corresponding to glutamic acid at position 64 of SEQ ID NO: 16 with glycine (G),
substitution of the amino acid corresponding to alanine at position 74 of SEQ ID NO: 16 with glycine (G),
substitution of the amino acid corresponding to phenylalanine at position 81 of SEQ ID NO: 16 with isoleucine (I),
substitution of the amino acid corresponding to leucine at position 86 of SEQ ID NO: 16 with isoleucine (I),
substitution of the amino acid corresponding to phenylalanine at position 87 of SEQ ID NO: 16 with valine (V),
substitution of the amino acid corresponding to glutamic acid at position 143 of SEQ ID NO: 16 with glycine (G),
substitution of the amino acid corresponding to leucine at position 188 of SEQ ID NO: 16 with glutamine (Q), and
substitution of the amino acid corresponding to glutamic acid at position 267 of SEQ ID NO: 16 with valine (V).

13. The method of claim 9, wherein the mutant of CYP102A1 comprises amino acid substitutions that correspond to amino acid substitutions in the CYP102A1 of SEQ ID NO: 16, wherein said substitutions are selected from the group consisting of F87A, R47L/Y51F, A74G/F87V/L188Q, R47L/L86I/L188Q, R47L/F87V/L188Q, R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V, R47L/L86I/F87V/L188Q, R47L/F87V/E143G/L188Q/E267V, R47L/E64G/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/L188Q/E267V, and R47L/E64G/F81I/F87V/E143G/L188Q/E267V.

14. The method of claim 9, wherein the mutant of CYP102A1 comprises amino acid substitutions that correspond to amino acid substitutions in the CYP102A1 of SEQ ID NO: 16, wherein said substitutions are selected from the group consisting of A74G/F87V/L1188Q, R47L/L86I/L188Q, R47L/F87V/L188Q, R47L/F87V/L188Q/E267V, R47L/L86I/L188Q/E267V, R47L/L86I/F87V/L188Q, R47L/F87V/E143G/L188Q/E267V, R47L/E64G/F87V/E143G/L188Q/E267V, R47L/F81I/F87V/E143G/L188Q/E267V, and R47L/E64G/F81I/F87V/E143G/L188Q/E267V.

15. The method of claim 9, wherein said method is carried out in an in vitro system and wherein metabolites generated by the reaction do not inactivate the catalyst.