JP2000508163A - Monooxygenase cytochrome P450cam mutant - Google Patents

Abstract

  (57) [Summary] A monooxygenase cytochrome P450cam mutant in which the cysteine residue at position 334 has been removed.

Description

DETAILED DESCRIPTION OF THE INVENTION             Monooxygenase cytochrome P450cam mutant   The present invention relates to monooxygenase cytochrome P450cam variants.   Monooxygenases have activated or non-activated carbon-hydrogen bonds Catalyzes selective oxidation with oxygen¹And therefore potential uses in organic synthesis There is a great interest in the business. However, progress in this area has Isolates sufficient amounts of monooxygenase enzymes and / or related electron transfer proteins Has been hampered by the difficulty of doing so. More than 150 different cytochrome P45 Despite the availability of the amino acid sequence of monooxygenase 0, There are only three types of structure data available^2,3,4In bacterial systems Few have been successfully overexpressed^Five.   Certain cytochrome P450 monomers capable of expressing a soluble and sufficient amount Xygenase is a highly specific P-450cam from P. putida. This enzyme is used for the localization of camphor to 5-exo-hydroxy camphor. Catalyze selective and stereoselective hydroxylation⁶. High resolution of P-450cam The crystal structure of^TwoThe mechanism of action of this bacterial enzyme is the production of its counterpart in mammals. P-450cam is believed to be very similar to It is used as a basic framework for structural models of enzymes.   The nucleotide sequence of P-450cam and the corresponding amino acid sequence are described in the literature Has been^5,7. The location of the active site of this enzyme is known, and the relationship between structure and function is known. Entities are being studied^8,9. Mutants of P-450cam are 101, 185, At the 247th and 295th sites^9,10,11, And at the 87th site Things¹²Is described in the literature. Tyrosine at position 96 (Y96) is 9 A mutant that changed to phenylalanine at the sixth position (Y96F mutant) is described in the literature. Has been described^11,13,14,15. However, in all cases, the dissertation Oxidation reaction of a molecule previously indicated as a substrate for a wild-type enzyme It reports on its effects. How to use different variants of different substrates There is no teaching on whether to provide a biocatalyst for oxidation.   In an attempt to develop a new biocatalyst, the present inventors re-used P-450cam. Designed (redesigned) to be a substrate for wild-type proteins The purpose is to enable the efficient oxidation of organic molecules Planning started.   As shown in FIG. 1, the three-dimensional structure of P-450cam shows that the active site is Close contact with the hydrophobic group of Uno by van der Waals force (close van de r Waals contact). Camphor and 244th part Contact with the side chains of leucine at position 2, valine at position 247 and valine at position 295 Is important. The three aromatic residues (Y96, F87 and F98) are grouped together Grouped and arranged in a substrate binding pocket, 9 There is a hydrogen bond between the sixth tyrosine and the carbonyl carbon of camphor. This It maintains the substrate in the correct orientation and ensures regio- and stereospecificity of the reaction.   Lipscomb, etc.¹⁶Showed that wild-type P-450cam dimerizes in 1978. Of the monomer and dimer for camphor oxidation. It also reports that the vehicle activity could not be identified. Dimerization reaction is thio The dimerization reaction can be reversed by an intermolecular cysteine It was concluded that this was caused by disulfide (SS) bond formation. They dimerize Is associated with one or more cysteines per P-450cam molecule I couldn't. In addition, the key cysteine residue involved in this reaction was It could not be identified. This is because the amino acid sequence of P-450cam and And the crystal structure was unknown at this point.   We have used molecular modeling to identify active Three aromatic residues in the active site pocket (Y96, F87, F98) were studied. The present inventors, Substituting any of these aromatic residues with a small hydrophobic non-aromatic side chain results in more "Aromatic pocket" that can be used to bind hydrophobic substrates (Aromatic pocket) ". program GRID¹⁷Using an aromatic probe and the aforementioned residue changed to alanine Possible variants of Rom P-450cam (F87A, Y96A and F98A ) Was calculated. Then the results are Cage Quanta¹⁸Was examined graphically.   Mutant F98A is located within the active site cavity accessible to the aromatic probe. Has a strong binding interaction, slightly smaller for Y96A and substantially smaller for F87A. Was found to be less. In the first example, the 96th It was decided to mutate the tyrosine at the site to alanine. Because the binding poke Because it is more central to the On the other hand, the 98th site Of phenylalanine is present in the groove for a surface. Also, the 96th site Removal of tyrosine will reduce substrate specificity for camphor. why If so, the hydrogen bond to the substrate is lost.   According to one aspect of the present invention, a monooxygen having the cysteine residue at position 334 removed. Mutants of the enzyme cytochrome P450cam are provided.   Preferably, the removal removes the cysteine residue from another amino acid other than the cysteine residue. Is performed by substituting   Alternatively, removal is by removing the entire 334th cysteine residue from the enzyme. Is performed.   If necessary, replace the tyrosine residue at position 96 in the mutant with another residue other than tyrosine. Replace with   Conveniently, the amino acids are the following amino acids: alanine, arginine , Asparagine, aspartic acid, cysteine, glutamic acid, glutamine, Lysine, histidine, isoleucine, leucine, lysine, methionine, proline , Serine, threonine, tryptophan, tyrosine and valine. Selected. However, when the position 334 is a cysteine residue, The acid is not cysteine and if the 96th site is a tyrosine residue, Noic acid is not tyrosine.   Preferably, 87, 98, 101, 185, 193, 244, 247, 295 Amino acids at one or more of positions 297, 395 and 396 are different Has been substituted.   The present inventors have proposed a published crystallographic atomic configuration. Co-ordinate) created P-450cam structure by modeling program   Investigated using Quanta. We present near the P-450cam surface Is involved in intermolecular disulfide bond formation leading to protein dimerization? 5 possible cysteines (58, 85, 136, 148 and 334) Stain) was determined. The present inventors performed site-directed mutagenesis, These cysteines were replaced with alanine, and five Cys-Ala surface mutants (surfac emutant).   Tamper in wild-type P-450cam and 5 Cys-Ala surface mutants The extent of dendrimerization was studied. Wild-type P-450cam and C58A , C85A, C136A and C148A mutants, the presence of dimers was Anion exchange high speed protein solution in Resource Q column (Pharmacia) Body chromatography (anion exchange fast protein liquid chromatography ) And Superose 12 column (Pharmacia) Exclusion chromatography. On the other hand, the C334A mutant Therefore, no dimer is detected, even at high concentrations (in the range of 0.1 mM). won. We have found that wild-type P-450cam is one of two protein molecules. Due to the formation of an intermolecular SS disulfide bond between the surface cysteine at position 334, It was concluded that it would undergo multimerization.   The C334A mutant clearly eliminates undesired protein dimerization. Has a white benefit and therefore the presence of single species in solution. Always guarantee your presence. In addition, we note the completely unexpected benefit of this variant. I saw it. Like all proteins, wild-type P-450cam is in a stationary state. Indicates aggregation. It is not clear why the protein aggregates, but P-450 The cam aggregate is insoluble and catalytically inert. Wild type The C58A, C85A, C136A and C148A mutants were all Aggregation and dimerization even in storage at -20 ° C and 50% glycerol solution Showed embodiment. Agglomeration during turnover, especially any commercially viable High concentration of P-450cam required for industrial use, especially for organic molecule synthesis It will happen no matter what. The C334A mutant, even at a concentration of mM, Over time, they did not show any evidence of aggregation. Therefore, C334 The A variant is useful for protein handling, storage and increased catalyst shelf life. Has an advantageous effect.   The present inventors have found that the mutation at the 96th site indicates that the mutated enzyme Are believed to be the key to enabling catalysis of organic substrates. Active site of enzyme Other amino acids adjacent to are used to alter the shape and specificity of the active site It may be mutated. These other amino acids include 87, 98, 101, 185 193, 244, 247, 295, 297, 395 and 396 Contains amino acids. Amino acids at one or more of these sites are Substitution with small hydrophobic amino acids to extend the active site, or shrink the active site Substituted with large hydrophobic amino acids to interact with the aromatic ring of the substrate To be replaced by an amino acid having the corresponding aromatic ring I have.   For the oxidation reaction, the conditions are described in the literature in the attached reference list . Enzyme systems are typically putidaredoxin (putidaredoxin) in addition to the mutated enzyme. xin) and includes putidaredoxin reductase with NADH as a cofactor In. Examples of cyclohexylbenzene oxidation can be found in the experimental section below. Has been described. Various classes of organic compounds are foreseen and described below. The present inventors have reported that wild-type P-450cam is not Note that it is active against oxidation of a number of molecules. However, all In some cases, the mutant P-450cam protein has a very high turnover -Shows activity.   i) organic compounds do not inactivate or denature aromatics, hydrocarbons or enzymes Any of the compounds used under the conditions. Mutations occur in the active site of the enzyme Mutated enzymes are used to create an aromatic-binding pocket Can catalyze the oxidation of aromatic compounds. Aromatic and polyaromatics as examples Oxidation of aromatic compounds is described in the experimental section below. Surprisingly, the wild-type enzyme is It has been reported to catalyze the oxidation of only Millie members, but some others Molecule of, for example, styrene¹⁹, Ethylbenzene^9,10, Tetralone derivative²⁰And Nico Chin^{twenty one}Etc. showed low activity.   ii) The organic compound is a hydrocarbon, for example, an aliphatic or alicyclic compound having a functional group. (See Scheme 1). Aromatic protecting groups are converted to functional groups before the oxidation reaction Addition and removal from the functional groups after the oxidation reaction. A suitable aromatic group is a benzyl group . Protecting groups serve two purposes. One is to make the substrate more hydrophobic, Increasing binding to hydrophobic enzyme pockets. Second, the substrate is the active site That may help to keep. Therefore, right The use of aromatic protecting groups allows the regioselective and stereoselective hydroxylation of the substrate. Silation will be achieved. Examples of monofunctionalized hydrocarbons Examples include cyclohexyl, cyclopentyl, and alkyl derivatives (salts). Chiem 1). The oxidation products of these compounds are converted into organic compounds, especially in homochiral form. It is a valuable starting material when formed in Foreseeable range of aromatic protecting groups Such as benzyl or naphthyl ether and benzoyl ether and (Scheme 1). Benzoxazole groups as carboxyl protecting groups and Benzyl oxazolidine groups as aldehyde protecting groups are of interest. Both Can be easily cleaved after enzymatic oxidation and produce aldehydes and acids Described in the literature on oxidation^{twenty two}.   iii) The organic compound is a C4-C12 aliphatic or alicyclic hydrocarbon. Shi The oxidation of cyclohexane and linear and branched hydrocarbons is described in the experimental section below. Has been demonstrated in The present inventors have also determined that wild-type P-450cam Can oxidize the offspring, but its activity is low, and the mutant It was found to show substantially high activity.   iv) The organic compound is a halogenated aliphatic or cycloaliphatic hydrocarbon. Linden ( The oxidation of hexachlorocyclohexane) is also described below.   Active site substitution and surface mutation of cysteine at position 334 to alanine (Surface mutation) and tyrosine at the 96th site Containing alanine, leucine, valine or phenylalanine instead of (Y96) Were constructed. Finally, some active site mutations (active site mutation) and a surface mutation to construct a mutant enzyme having multiple mutations. Was. Cytochrome P-450cam, its natural electron transfer partner, Putida The genes encoding redoxin and putidaredoxin reductase were Using polymerase chain reaction (PCR) from total cellular DNA of P. Putida And amplified. The combination of expression vector / E. Coli host used was , P-450cam is pRH1091 in the JM109 strain.^{twenty three},Petit Daredoxin is pUC118 in the JM109 strain, The reductase was pGL W11 in the DH5 strain. Oligonucleotide Oligonucleotide-directed site-specif ic mutagenesis), the method of Zoller and Smith^{twenty four}According to M13mp19 Making a Loan and Selecting Mutants by Kunkel's Method^{twenty five}Was performed.   The binding of potential substrates was studied by spectroscopic methods. In the absence of substrate Wild-type enzyme is a 6-co-ordinated low-spin form and is weak Bound water occupies the sixth coordination site and is characterized by a maximum Soret band at 418 nm (Soret maximum). Amantanone, which is camphor and a substrate analog, The binding of adamantane and norbornane binds heme with the characteristic sole at 392 nm. It completely converted to a 5-coordinated high spin form with a negative band. The spin state of this hem The shift (haem spin-state shift) is achieved by increasing the reduction potential of heme. Which indicates that the physiological electron transfer partner, putidaredoxin, has P -450 cam can be reduced to initiate the catalytic hydroxylation cycle. And enable²⁶. Therefore, the spin state shift of heme is shown in Tables 1 and 2. As can be seen, the molecules oxidized by the wild-type and P-450cam enzyme variants It is a qualitative sign of prospect.   Buffer (50 mM, Tris-HCl, pH 7.4) containing 10 μM Toxin, 2 μM putidaredoxin reductase, 1 μM cytochrome P-450c am monooxygenase (wild type or mutant), 200 mM KCl, 50 μg / Ml bovine liver catalase (Sigma) and 1 mM target organic compound such as Buffer containing rohexylbenzene (added as a 0.1 M stock in ethanol) The solution, typically 3 ml, was preincubated at 30 ° C. for 5 minutes. NADH all The enzymatic reaction was started by adding to a concentration of 2 mM. 4 additional Aliquots of NADH (to increase the NADH concentration by 1 mM each time) Add at 10 min intervals, incubate for 30 min, aliquot of one substrate (To increase the concentration by 1 mM) was also added. After 60 minutes elapse, chloroform The reaction was terminated by adding 0.5 ml of the solution and vortexing the mixture. Was. The layers were separated by centrifugation (4000 g) at 4 ° C. Chloroform The layers were analyzed by gas chromatography.   P-450cam-mediated oxidation products are commercially available For a number of, but not limited to, substrate compounds such as cyclohexylbenzene, The roform extract was evaporated to dryness under a stream of nitrogen. Hexane residue And oxidized products were separated by high performance liquid chromatography. Elution using a 1 / isopropanol gradient. The purified product is transferred to a mass spectrometer. And especially by nuclear magnetic resonance spectroscopy.   For different substrates with different solubilities in aqueous buffers, incubate The substrate mass added to the plate mixture was varied between a final concentration of 0.2-0.4 mM. The NADH concentration was monitored at 340 nm and in all cases the incubation was Additional substrate and NADH were added during the reaction.   Using the experimental techniques described above, we have determined that wild-type P-450cam and mutant A significant number of organic compounds were studied as substrates for both body Y96A. Laboratory Ultimately, mutant: Y96V; Y96L: Y96f: C334A: combined ) Mutant: F87A-Y96G-F193A and complex mutant of active site and surface : Y96A-C334A, Y96L-C334A, Y96F-C334A, F87A-Y96G-F193A-C334A. C334A and C The results for 334A-Y96A are shown in Tables 1 and 2. Here, structurally Related molecules are grouped together.   Table 1 shows that small linear, branched and cyclic carbonizations by mutant Y96A-C334A. It lists the consumption of NADH for the oxidation of hydrogen. Tables 2 (a) to 2 (h) Product distributions for mutant and substrate combinations identified to date They are listed.   The cysteine residue at position 344 is well known and freely available. Can be removed by standard restriction techniques that can Will not be explained.Scheme 1: ^aThe values were determined using the method of Sligar (S.G. Sligar, Biochemistry. 1976.15.5 5399-5406). Average of two independent measurements used. K_appIs the value of K in the buffer.⁺Concentration of Heavily dependent on [K⁺]> 150 mM, K to camphor_app Is 0.6 μM for both wild type and Y96A. The data in this table are high To avoid salting out of the substrate at the ON concentration, in a phosphate buffer (pH 7.4): [K⁺] = 70 mM.^b Saturation was not reached.                                      Table 3     Turnover of small alkanes by P450cam mutant     All variants listed below contain the C334A mutation.         Turnover speed measured as NADH consumption speed         (Nanomolar NADH / nanomolar P450cam / sec)Structure and analysis of the product following oxidation of the substrate by the P450cam active site mutant cloth "Background"-a typical background NADH oxidation rate of 0.0 7 nanomolar NADH (nanomolar P450cam)^-1Second^-1It is.

[Procedure amendment] [Submission date] May 1, 1998 (1998.5.1) [Correction contents] The scope of the claims 1. A monooxygenase in which the cysteine residue at position 334 has been removed Cytochrome P450cam mutant. 2. Removal is achieved by replacing a cysteine residue with an amino acid other than cysteine. 2. The mutant according to claim 1, which is carried out. 3. Removal is by removing the entire cysteine at position 334 from the enzyme. 2. The mutant according to claim 1, which is carried out. 4. The tyrosine residue at position 96 in the mutant is another amino acid other than tyrosine The mutant according to any one of claims 1 to 3, which is substituted with: 5. The amino acids are alanine, arginine, asparagine, aspartic acid, glue Tamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine Methionine, proline, serine, threonine, tryptophan, tyrosine and 5. The method of claim 1, 2 or 4 selected from the group consisting of A variant according to any of the preceding claims. 6. 87, 98, 101, 185, 193, 244, 247, 295, 297, The amino acid at one or more of the positions 395 and 396 is another amino acid The mutant according to any one of claims 1 to 5, wherein the mutant is substituted with a residue.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM , AU, AZ, BB, BG, BR, BY, CA, CN, CZ, EE, GE, HU, IL, IS, JP, KE, K G, KP, KR, KZ, LK, LR, LS, LT, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, RO, RU, SD, SG, SI, SK, TJ, T M, TR, TT, UA, UG, US, UZ, VN (72) Inventor Nickerson Darren Paul             United Kingdom Oxford Oeck             S1 3U Manor Road             Leewell Manor (No street address) (72) Inventor Hart Alwin James             United Kingdom Leicestershire EL11             3 Cubie Roughborough Wood Brook             Claude 33

Claims (1)

[Claims] 1. A monooxygenase in which the cysteine residue at position 334 has been removed Cytochrome P450cam mutant. 2. Removal is achieved by replacing a cysteine residue with an amino acid other than cysteine. 2. The mutant according to claim 1, which is carried out. 3. Removal is by removing the entire cysteine at position 334 from the enzyme. 2. The mutant according to claim 1, which is carried out. 4. The tyrosine residue at position 96 in the mutant is another amino acid other than tyrosine The mutant according to any one of claims 1 to 3, which is substituted with: 5. The amino acids are alanine, arginine, asparagine, aspartic acid, glue Tamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine Methionine, proline, serine, threonine, tryptophan, tyrosine and Any one of claims 1, 2 and 4 selected from the group consisting of A variant according to any of the preceding claims. 6. 87, 98, 101, 185, 193, 244, 247, 295, 297, The amino acid at one or more of the positions 395 and 396 is another amino acid The mutant according to any one of claims 1 to 5, wherein the mutant is substituted with a residue. 7. What is substantially described above in connection with the accompanying drawings and / or examples An oxygenase cytochrome P450cam mutant.