SK55598A3 - Mutant mono-oxygenase cytochrome p450cam - Google Patents

Abstract

A mutant of the mono-oxygenase cytochrome P450cam in which the cysteine residue at position 334 is removed.

Description

Technical field

The invention relates to a mutant form of cytochrome P-450 monoxygenase.

BACKGROUND OF THE INVENTION

Monooxygenase catalyzes the selective oxidation of activated and non-activated carbon-hydrogen bonds with oxygen, see de Montellano OPR and the editor of Cytochrome P-450: Structure, Mechanism, and Biochemistry, Plenum Press, New York, 1986. For this reason, it is also an enzyme with potential of great importance for organic synthesis. The use of the enzyme in this field has hitherto been hampered by the difficulty of obtaining a sufficient amount of self-monoxygenase and / or obtaining associated protein electron transporters. Although the amino acid sequences of more than 150 different cytochrome P-450 monoxygenases are available, structural data of only three of them are still available, see Poulos T.

L., Finzel B. C. and Howard A. J., J. Mol. Biol., 1987, 195, 687-700; Hasemann C.A., Ravichandran K.G., Peterson J.A. and Deisenhofer J., J. Mol. Biol. 1994, 236, 1169-1185; Ravichandran K.G., Boddupalli S.S., '

Hasemann C.A., Peterson J.A. and Deisenhofer J., Science, 1993, 261, pp. 731-736. Some of these enzymes have been successfully expressed in bacterial systems, see Unger B. P., Gunsalus I. C. and Sligar S. G., J. Biol. Chem., 1986,

261, pages 1158 to 1163; Miles J. S., Munro k. VI., Rospendowski B. N., Smith W. E., McKnight J. and Thomson A. J., Biochem. J., 1992, 288, pp. 503-509; Richardson T. H., ϊ

Hsu Μ. J., Kronbach T., Bames H. J., Chan G., Waterman M.

R., Kemper B. and Johnson E. F., Ar. Biochem. Biophys.

1993, 300, pp. 510-516; Boddupalli S.S., Oster T., Estabrook R.W. and Peterson J.A., J. Biol. Chem., 1992, 267, 10375-10380; Li H. ', Darish K. and Poulos T. L., J. Biol. Chem., 1991, 266, pp. 11909-11914.

One of the cytochrome P-450 monoxygenases that is soluble and can be expressed in sufficient amounts is the highly specific P-450cam of P. putida, which catalyzes the site-specific and stereo-selective hydroxylation of camphor to 5-exo-hydroxy camphor, see Gunsalus IC and Wagner, GC, Methods Enzymol., 1978,

52, pages 166-188. The high resolution crystal structure of P450cam was determined, see Poulos T. L. et al; J. Mol. Biol. Since a very similar mechanism of action of this bacterial enzyme as well as its mammalian counterpart is envisaged, this bacterial enzyme has been used as the basis on which the structural models of mammalian enzymes are based.

The nucleotide sequence and the P-450cam amino acid sequence derived therefrom have also been described, see Unger BP et al., J. Biol. Chem., 1986; Miles JS et al; Biochem. J., 1992; Richardson TH et al., Ar. Biochem. Biophys., 1993; Boddupalli SS et al., J. Biol. Chem., 1992; ¹ Li H. et al., J. Biol. Chem., 1991; Haniu M., Armes LG, Yasunobu KT, Shastry BA and Gunsalus IC, Biol. Chem., 1982, 257, pages 12664-1267. Localization of the active site of the enzyme is also known, and relationships between its structure and function are also known. Mutant forms of P-450cam have also been described, with mutations at positions 101 and 185 and at positions 247 and 295, see Loida PJ and Sligar SG, Biochemistry, 1993, 32, 11530-11538; Loida PJ and Sligar SG, Protein Eng., 1993, 6, 207-212; Atkins WM and Sligar SG, J. Am. Chem. Soc., 1989, 111, pp. 2715-2717; and at position 87, see Tuck SF, Graham-Lorence S., Peterson JA and Ortiz de Montellano PR, J. Biol. Chem., 1993, 268, pp. 269-275. A mutant form of this enzyme has also been described wherein the tyrosine at position 96 (Y96) has been replaced by phenylalanine 96 (mutant Y96F), see Atkins WM and Sligar SG, J. Am. Chem. Soc., 1989, 111, pp. 2715-2717; Di Prime C., Hui Bin Hoa G., Douzou P. and Sligar SG, J. Biol. Chem., 1990, 265, pp. 5361-5363; Atkins WM and Sligar SG, J. Biol. Chem., 1988, 263, pp. 18842-18849; Atkins WM and Sligar SG, Biochemistry, 1990, 29, pages 1271 to 1275. In all cases, the effects of mutations on the oxidation reactions of molecules that were known earlier as substrates for the wild-type enzyme are described.

However, it is not known in the art how mutant forms of the enzyme can be used as biocatalysts to oxidize other, novel and unusual substrates.

In an effort to develop new biocatalysts, a project was proposed to alter P-450cam so that the enzyme was able to efficiently carry out specific oxidations of organic molecules independently of whether or not they are substrates for the naturally occurring form of the enzyme.

The three-dimensional structure of the P-450cam has an active site that is in close contact with the hydrophobic groups of the camphor by means of van der Waals forces (see Figure 1). Of particular importance are the interactions between the camphor and the side chains of leucine 244, valine 247 and valine 295. Three aromatic residues (Y96, F87 and

F98) are grouped to flank the substrate binding pocket together with the hydrogen bond between the tyrosine 96 and the carbonyl oxygen of the camphor to maintain the substrate in the correct orientation to ensure proper positioning and stereo-specificity of the reaction.

Lipscomb and coworkers (see Lipscomb JD, Harrison JE, Dus K.M. and Gunsalus I.C., Biochem. Biophys.

Res. Commun., 1978, 83, pp. 771-778) in 1978 showed that the naturally occurring form of P-450cam tends to dimerize, but further states that the catalytic activity of monomeric and dimeric enzymes in relation to camphor oxidation cannot be distinguished. . Since dimerization can be reversed using thiol group reducing agents, the authors conclude that dimers are formed by intermolecular cysteine disulfide bridges (S-S). However, the authors have not been able to determine whether dimerization is involved in one or more cysteines in a single P-450cam molecule. They were also unable to identify the cysteine residue (or residues) involved in this reaction, since neither the amino acid sequence nor the crystal structure of P-450cam was known at that time.

Molecular modeling was used to elucidate the likely effects of point mutations of the three aromatic amino acid residues (Y96, F87 and F98) in the enzyme active site pocket. Replacing any of these aromatic residues with smaller hydrophobic, non-aromatic side chains results in an " aromatic pocket that could bind more hydrophobic substrates. The GRID program was used to calculate the energy of the interaction between the aromatic probe and possible cytochrome P-450 monoxygenase mutants where the alanine residues (F87A, Υ96Α and F98A) were listed, see Goodford P.J., J. Med. Chem., 1985, 28, 849-857. The results were further graphically processed using the Quanta software package (Quanta 4.0, Molecular Simulations Inc., 16 New England Executive Park, Burlington, MA 01803-5297) for molecular modeling.

The F98A mutant appears to exhibit the strongest binding interaction at the active site available for the aromatic probe, while for Y96A this interaction is slightly weaker and for the latter F87A mutant is significantly lower. For the first attempts, it was decided to replace tyrosine 96 with alanine, since it is located closer to the center of the binding pocket, while phenylalanine 98 is located closer to one of the edges. Also, removal of tyrosine 96 appears to reduce the specificity of the enzyme to camphor, due to the loss of the possibility of hydrogen bonding to the substrate.

SUMMARY OF THE INVENTION

The invention provides a mutant form of cytochrome P-450cam monoxygenase wherein at position 334 the cysteine residue is removed.

In a preferred embodiment of the invention, the removal is accomplished by substituting a cysteine residue with any other amino acid (except cysteine).

In another embodiment of the invention, cysteine residue 344 can be removed by deletion of this amino acid residue from the enzyme.

In another embodiment of the invention, the tyrosine residue at position 96 in mutant form can also be replaced by any amino acid residue other than tyrosine.

In a preferred embodiment, the amino acid suitable for substitution may be selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, tryptophan, threon, threonine tyrosine and valine, provided that cysteine at position 334 cannot be a replacement amino acid for cysteine, and tyrosine at position 96 cannot be a replacement amino acid for tyrosine.

In another preferred embodiment of the invention, the amino acid residue at one or more of the following positions: 87, 98, 101, 185, 193, 244, 247, 295, 297, 395 and 396 is replaced by another amino acid residue.

Using the Quanta modeling program, the structure of the P-450cam created from published crystallographic atomic coordinates was investigated. It has been found that there are five cysteines near the surface of the P-450cam (cysteines 58, 85, 136, 148, 334) that can participate in the formation of intermolecular disulfide bonds, which then lead to the formation of protein dimers. Specific mutagenesis was performed locally so that each of the above cysteines was substituted with alanine. This resulted in five mutant forms of the enzyme with a " Cys-Ala surface mutation (" surface mutants).

The extent of protein dimerization occurring in the wild-type P-450cam and in the five "surface mutants of Cys-Ala" was investigated. The presence of the dimer was determined by anion exchange fast chromatography on a Resource-Q column (Pharmacia) as well as by gel filtration on a Superose 12 molecular sieve column (Pharmacia) for the naturally occurring P-450cam type and for the mutant forms C58A, C85A, C135A and

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C148. No dimer was found in the C334A mutant form (see Figure 2) even at a high concentration (in the range of 0.1 mM). Thus, we conclude that naturally occurring P-450cam dimerizes by intermolecular disulfide bonds

S-S between surface cysteines 334 on two protein molecules.

The mutation at position C334A has a clearly visible effect on the removal of unwanted protein dimerization, only monomeric enzyme molecules were always present in the solution. Furthermore, a completely unexpected further advantageous feature of this mutation was noted. Like all proteins, naturally occurring P450cam exhibits aggregation of molecules after some time. The reason for protein aggregation is unclear, but P-450cam aggregates are insoluble and catalytically inactive. Naturally occurring P-450cam, as well as mutant forms of C58A, C85A, C136A and C148A, dimerize during storage at 4 ° C and even when stored in 50% glycerol at -20 ° C and further on aggregation of molecules. Aggregation also occurs during the catalytic function of the enzyme, especially at higher concentrations of P-450cam, which are necessary for economically feasible industrial applications, such as the synthesis of organic molecules. The mutant form of C334A showed no aggregate formation, even when using millimole concentrations for three days and at room temperature. It can be concluded that the mutation at C334A has a beneficial effect on the handling, storage and prolongation of the catalytic effect of these proteins.

We believe that the mutation at position 96 is crucial for the ability of the mutated form of the enzyme to catalyze the oxidation of a relatively wide range of organic substrates. Other amino acids adjacent to the active site of the enzyme may also be mutated to alter the shape and specificity of the active site. These amino acids are residues at positions 87, 98, 101, 185, 193, 244, 247, 295, 297, 395, and 396. It is contemplated that amino acids at one or more of these positions may be replaced by either a small hydrophobic amino acid, thereby increasing the active site of the enzyme, or a large hydrophobic amino acid, resulting in a reduction in the size of the active site. Further, it may be replaced by an amino acid having an aromatic ring, which may interact with the respective aromatic ring of the substrate.

The conditions of the oxidation reactions are described in the accompanying references. A typical enzyme system usually still includes, together with the mutated form of the enzyme putidaredoxin and putidaredoxin reductase with NADH as cofactors. An example of cyclohexylbenzene oxidation is described below in the experimental section. An example of the various types of organic compounds contemplated is also described. We have found that the naturally occurring P-450cam is active in oxidizing the large number of molecules described in the following sections. However, in all cases, the mutated form of P-450cam had a much higher activity ¹ in substrate transformation.

(i) Organic compound means an aromatic compound, either a hydrocarbon or other compound used under conditions that do not inactivate or denature the enzyme. Because the mutation was made to create an aromatic-binding enzyme capsule in the active enzyme center, the mutated form of the enzyme is capable of catalyzing the oxidation of a wide variety of organic compounds. Examples of oxidation of aromatic and polyaromatic compounds are described below in the experimental section. The oxidation of these compounds is quite surprising given that it is known that the naturally occurring form of the enzyme is capable of oxidizing only compounds structurally derived from camphor and has little activity against several other molecules such as styrene, see Fruetet JA, Collins JR, Camper DL, Loew GH and Ortiz de Montallano PR, J. Jun. Chem. Soc., 1992, 114, 6987-6993; ethylbenzene, see Loida P.J. and Sligar S.G., Biochemistry, 1993, 32, 11530-11538; Loida P.J. and Sligar S.G., Protein Eng., 1993, 6, pp. 207-212, tetralone derivatives, see Watanabe T. and Ishimura Y. J., Jun. Chem. Sec. 1989, III, pp. 410-41; and nicotine, see Jones J P., Trager W. F. and Carlson T. J., J. Jun. Chem. Soc., 1993, 115, pages 381-387.

ii) The organic compound may also be a hydrocarbon such as an aliphatic or alicyclic hydrocarbon bearing a functional group (see Scheme 1). Prior to the actual oxidation reaction, the aromatic protecting group is bound to ¹ functional group and, after the oxidation reaction, (said protecting group) is removed from the functional group. A suitable protecting group is benzyl. Protecting groups serve two purposes - firstly, making the substrate more hydrophobic, thereby enhancing its binding to the hydrophobic pocket of the enzyme, and secondly, helping to keep the substrate in place at the active center of the enzyme. Thus, both site-specific and space-selective hydroxylation of the substrate can be achieved using suitable aromatic protecting groups. Examples of single-functional hydrocarbons are cyclohexane, cyclopentane and alkyl derivatives (see Scheme 1). The oxidation products of these compounds are a valuable starting material for organic syntheses, especially when produced in chiral homogeneous form. Protecting groups used include, for example, benzyl or naphthyl ethers or benzoyl ethers and amides (see Scheme 1). Also important are benzoxazole groups which are suitable as carboxyl protecting groups and N-benzyloxazolidine groups which serve to protect aldehyde groups. Both of these types of protecting groups can easily be removed after enzymatic oxidation and have been described in the literature on microbial oxidation of aldehydes and acids, see Davis HG, Green RH, Kelly DR and Roberts SM: "Biotransformation in Preparative Organic Chemistry, Academic Press, London" , 1989, page 169 ff.

iii) The organic compound is further a C 4 to C 12 aliphatic or alicyclic hydrocarbon. The oxidation of cyclohexane and linear and branched hydrocarbons is described below in the experimental part. It has been found that a naturally occurring form of the enzyme P-450cam can also be used to oxidize these molecules, but its activities are low and the mutant forms have substantially higher activities in all cases.

iv) The organic compound may also be a halogenated aliphatic or alicyclic hydrocarbon. The oxidation of lindane (hexachlorocyclohexane) is described below.

Enzyme mutants have also been created in which substitution at the active center is combined with a surface mutation. In these mutants, cysteine at position 334 is replaced by alanine and, instead of tyrosine at position 96 (Y96), contains alanine, leucine, valine or phenylalanine. Finally, several mutations in the active site and said mutation on the surface of the enzyme were combined to form mutant forms of the enzyme with multiple mutations. The genes encoding cytochrome P-450cam and its natural electron transfer partners putidaredoxin and putidaredoxin reductase were amplified using polymerase chain reaction (PCR) from total cellular DNA isolated from P. putida. Further, the following combinations of expression vector and E. coli host strain were used: vector pRH1091 (see Baldwin JE, Blackburn JM, Heath RJ and Sutherland JD, Bioorg. Med. Chem. Letts. 1992, 2, pp. 663-668). strain JM109 for P-450cam; vector pUC118 in strain JM109 for putidaredoxin; and the pGL W11 vector in strain DH5 for putidaredoxin reductase. Targeted oligonucleotide mutagenesis was performed using the M13 subclone following the procedure of Zoller and Smith (see Zoller, M.J. and Smith, M., Nucleic Acids Res., 1982, 10, 6487-6500). Mutant selection was performed by the method of Kunkel (see Kunkel T.).

A.) Proc. Natl. Acad. Sci. USA, 1985, 82, 488-492).

The binding of potential substrates was investigated by spectrophotometric methods. The naturally occurring enzyme occurs in a hexacoordinated low spin form with poorly bound water at the sixth coordination site and exhibits a characteristic Soret spectrum at a wavelength of 418 nm.

Binding of camphor and analogous substrates such as adamantanone, adamantane, and norbonane will change the heme ring to a pentacoordinated high-spin form having a characteristic Soret band at 392 nm. This shift in the heme spin state is accompanied by an increase in the heme reduction potential, which allows physiological transfer of electrons from putidaredoxin while reducing the P-450cam and initiating a catalytic hydroxylation cycle, see Sligar S.G. and Gunsalus I. C., Proc. Natl. Acad. Sci. USA, 1976, 73, pages 1078-1082. The shift of the heme spin state is such a qualitative indicator of the probability that the molecules listed in Tables 1 and 2 are oxidized by the naturally occurring type P-450cam, respectively by its mutant forms.

DETAILED DESCRIPTION OF THE INVENTION

Buffered solution (50mM Tris.HCl, pH 7.4), usually with a volume of 3 ml, containing ΙΟμΜ putidaredoxin, 2μΜ putidaredoxin reductase, tochμΜ cytochrome P-450cam monooxygenase (naturally occurring type or mutant form), 200 mM KCl, 50 µg / ml ml of bovine liver catalase (Sigma) and 1 mM target organic compound, such as cyclohexylbenzene (added as a 0.1M stock solution in ethanol) were incubated at 30 ° C for 5 minutes. The enzyme reaction was started by adding NADH to a final concentration of 2 mM. Four aliquots of NADH were added at 10 minute intervals to increase its concentration by 1 mM each. After 30 minutes of incubation, one aliquot of the substrate was added to increase its concentration by 1 mM. The reaction was stopped after 60 minutes by adding 0.5 ml of chloroform and vortexing the mixture. The phases were separated by centrifugation (4000 x g) at 4 ° C. The choroform layer was analyzed by gas chromatography.

With respect to a number of substrate compounds, such as cyclohexylbenzene, for which not all P-450cam oxidation products are commercially available, the chloroform extracts are evaporated to dryness under a stream of nitrogen. After evaporation, the residues are extracted with hexane and the oxidation products are separated by high pressure liquid chromatography, eluting with a hexane-isopropyl alcohol gradient. The purified products are then identified by mass spectroscopy and in particular by magnetic resonance spectroscopy.

For different substrates, which differ in their solubility in aqueous buffer, the amount of substrate added to the incubation mixtures varies. The concentration varies from 0.2 to 4 mM of the final substrate concentration. The NADH concentration is measured by spectrophotometry by absorbance at 340 nm, in all cases an additional amount of substrate and NADH were added during incubation.

Using the above experimental methods, the inventors screened a large number of organic compounds that could serve as substrates for both the naturally occurring form of P-450cam and the mutated version of the Y96A enzyme. The invention encompasses mutants designated Y96V, Y96L, Y96f, C334A, the combined mutant F87A-Y96G-F193A, and mutants with a combination of mutations at the active site and on the surface of the enzyme Y96A-C334A, Y96V-C334A, Y96L-C334A, Y96F-C334A and F87A- -F193A-C334A. The results for the mutant forms C334A and C334A-Y96A are summarized in Tables 1 and 2, where structurally similar molecules are listed in common groups.

Table 1 describes the consumption of NADH for the oxidation reaction of small linear, branched, and cyclic hydrocarbons using the mutated form of the enzyme Y96A-C334A. Tables 2 (a) to 2 (h) describe the details of the distribution of the products of each mutant enzyme form and the appropriate substrate combinations for these enzymes where these data are known.

The cysteine residue at position 344 can be removed by porn from all freely available standard methods known in the art, and therefore its removal is not described in detail here.

Scheme 1

Table 1

Kapp (μΜ) ³ WT Y96A '7 1 6.3 12 2 12 28 & 3 s I 1.4. 4 330 92 cro 5 > 1500 ^p 73

^and Values are the average of two independent measurements using the Sliger method (Sligar SG, Biochemistry, 1976, 15, pp. 5399-5406). The Kapp value greatly depends on the concentration of K ⁺ ions in the buffer. At [K *] values> 150 mM, the Kapp for camphor is 0.6 μΜ for both the wild-type enzyme and the Y96A mutant. The data in this table was determined at [K ⁺ ] = 70 mM in phosphate buffer pH 7.4 to avoid salting out of the substrates at higher ion concentrations.

^b Saturation not reached.

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Conversion of alkanes by P450cam mutants

All mutants listed below also contain the C344A mutation. The conversion rate was determined as the loss of NADH (nmol NADH / nmol P450cam / s).

substrate Length of main chain Title Wild type Y96A C4 n-butane - - C4 2-methylbutane background 4, 6 C4 2,3-dimethylbutane background 16, 8 C4 2,2-dimethylbutane background 14.0 C5 n-pentane background 5, 8 C5 2-methylpentane 3, 8 11.7 C5 3-methylpentane 1.3 14.2 C5 2,4-dimethylpentane 0.2 12, 6 C5 2,2-dimethylpentane 5.2 12, 8 C5 2,2,4-trimethylpentane 0.9 5, 3 C5 3-ethylpentane background 16.2 C6 n-hexane background 6, 0 C6 2-methylhexane background 10, 6 C7 n-heptane 2.7 4.4 C7 2-methylheptane background 2.1 C7 4-methylheptane 1.4 10.2 C8 n-octane background 5, 8 C7 cycloheptane 4.4 42, 5

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Total quantity of products (area / 10 ⁵ ) p -9 r with • D or L L or d Cyclohexylbenzene products 1: cis-4-ol trans-4-ol I 3-ol ω 1 o t— ¹ about 00 12 25 I_ 20 43 WT Percentage of products for each mutant: -J ►B. 45 15 20 20 > - < within cn H ¹ K 13 σ> 27 ⁵⁴ Y96F 10, 4 16 23 23 38 The within σ> tr * 12, 5 23 10 ω Ό 28 The VO ch <

Table 4 (a)

Product structure and functional group deposition after oxidation of substrates using P450cam active site mutants.

All of the mutants shown below also contain the C334A mutation.

chemically reactive sites

Table 4 (b)

Total quantity of products (area / 10 ⁵ ) O ^: o 0 0 ^ Naphthalene products N) 1 ABOUT H * h- » 1 about M about • x about M en ABOUT H * ABOUT ABOUT WITH H 1 Percentage of products for each mutant: 1.1 ABOUT t-> ABOUT ABOUT The what en > NJ (Fc. ABOUT t- ¹⁰ O The CD ch about > -xj ABOUT J— ¹ o h; WHAT ch F M • x (Fc. ABOUT I- * about about The WHAT ch < ABOUT H ABOUT —— ¹ O O 00 hl to Are you g3 ch ABOUT 1

Table 4 (c)

►u tr <

CL h

o x

i— * o <4 OJ 3 0 '

Ό h

about

CL ct

Total quantity of products (Area / 10j σ about w > Products of phenanthrene 0, 075 35 12 ¹⁵ 38 WT Percentage of products for each mutant: j » about 15 13 23 49 Y96A cn 23 tn 31 41 The IX) cn hJ > 00 l-> <_n <X> 41 35, 5 Y96L t - ¹ * »<Ti. 10 11 38 x M Y96V- ABOUT ABOUT cn <J1 29 w 41 27 F87A-F96G- F193A

Table 4 (d)

fluoranthene

rt

0)

P!

rt

t)

Cn o

to d

ABOUT

X

K o

*with

OJ n

n> '• o l-i o

and c

x * rt ► <

Total quantity of products (area / 10) w Fluoranthene products about about about WITH H ►ti h about it •with -J t - ¹ m 00 h; Ό ch > adducts (c 1 I 1 Y96F Id D ABOUT m P r * p. ABOUT 1 T T The WHAT ch tti D n φ _j. (D d- P 1 1 1 Y96V- 0 rt H · H R + about > it l-d about about about ►ti WHAT ►ti to? Q 1 P P rt • ·

Table 4 (e)> Ό

The

WITH

C rt

OJ

P rt · <

►u

Cn

O (0 0) hydroxylated products

Total quantity of products (area / 10 °) about about the Pyrene products about about about about about WITH H The about M X * M H * b- ¹ ►Ch. cn ife. ω cn RFK about ► < CD <n > D α rt σ ) - ¹ CH M Cn X cn H * N) cn N) CD will give GJ CD cn CD M n Π) P a + CD ' ABOUT & b- » (J1 > » cn CD cn Cn it ca) ► < CD cn · t- P The Φ -Ι- Ο and P b- ⁱ cn WHAT H * N) cn Cn ω about The CD cn < ABOUT Γ + H H · WITH- ! rt about about t \) tO ABOUT about ca) ca) 00 hRL M f CD M Ca) > S cn ABOUT 1 CD P rt T:

Table 4 (f)

32Hexachlórcyklohexán

ie u cn

P u

ABOUT

w Ω 3 H ABOUT tn Rt. • P i-: (D 3 about 3 about H WITH and H TJ ui ABOUT 1 H * W 1 w Products of lindane (hexachlorocyclohexane) WITH tn 100 are you H Percentage of products for each mutant: (b ω • w Ui t - ¹ o o The CD ch

Table 4 (g)

Table 4h

Products derived from hexane Representation of products (%) for mutants Y96F Y96A 2-hexanone 10 15 3-hexanone 16 28 2-hexanol 24 26 3-hexanol 50 32 Relative activity (naturally occurring enzyme = l) 18.2 25, 5

Products derived from 2-methyl- hexane Representation of products (%) for mutants Y96F Y96A 2-methyl-2-hexanol 72 74 5-methyl-2-hexanone 16 14 2-methyl-3-hexanol 7 4 5-methyl-2-hexanol 5 8 Relative activity (naturally occurring enzyme = l) 2.3 2.6

FIGURE 1

V247 L244

ABOUT

O5

FIGURE 2 after injection (mini

Relative intensity

? ¾ P S5-36

Claims (7)

A cytochrome P-450cam monooxygenase mutant, characterized in that the cysteine residue at position 334 is deleted. ' 2. The mutant of claim 1, wherein removal of said cysteine residue is accomplished by substitution with an amino acid other than cysteine. The mutant of claim 1, wherein removal of said cysteine residue from the enzyme is accomplished by deleting the cysteine at position 334. Mutant according to any one of the preceding claims, characterized in that in this mutant the tyrosine residue at position 96 is replaced by any amino acid other than tyrosine. A mutant according to any one of claims 1, 2 or 4, wherein said amino acid is selected from the following group: alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine and valine. The mutant of any one of the preceding claims, wherein the amino acid residue at one or more of positions 87, 98, 101, 185, 193, 244, 247, 295, 297, 395, and 396 is replaced by another amino acid residue. The cytochrome P-450cam monooxygenase mutant, described above, with reference to the accompanying drawings and / or examples.