Field of the invention

The present invention relates to the use of a cytochrome P450 enzyme for catalysing the hydroxylation of organic substrates.

Background of invention

Cytochrome P450 (CYP) is a superfamily of haem-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide bound species at 450 nm. They are found in all kingdoms of life such as animals, plants, fungi, protists, bacteria, archaea, and furthermore a putative P450 from giant virus A. polyphaga has been recently proposed, Lamb, DC; Lei, L; Warrilow, AG; Lepesheva, Gl; Mullins, JG; Waterman, MR; Kelly, SL (2009). "The first virally encoded cytochrome P450". Journal of Virology. 83 (16): pp8266-9. Cytochrome P450 have not been identified in E.coli, Roland Sigel; Sigel, Astrid; Sigel, Helmut (2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley. ISBN 0-470-01672-8; Danielson PB (December 2002). "The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans". Curr. Drug Metab. 3 (6): pp561-97.

Cytochrome P450 shows extraordinary diversity in their reaction chemistry supporting the oxidative, peroxidative and reductive metabolism of a diversity of a range of endogenous and xenobiotic substrates.

In humans, cytochrome P450 are best known for their central role in phase I drug metabolism where they are of critical importance for two of the most significant problems in clinical pharmacology: drug interaction and inter- individual variability in drug metabolism.

The most common reaction catalyzed by cytochromes P450 is a mono- oxygenase reaction. Cytochrome P450 mono-oxygenases use a haem group to oxidise molecules, often making them more water-soluble by either adding or unmasking a polar group. In general the reaction catalysed by these enzymes can be summarised as:

R-H + 0 ₂ + 2e ® R-OH + H ₂ 0

Where R-H is the substrate and R-OH is the oxygenated substrate. The oxygen is bound to the haem group in the core of the CYP enzyme, protons (H ⁺ ) are usually derived from the reduced cofactor NADH or NADPH through specific amino acids in the CYP enzyme. CYP enzymes can receive electrons from a range of redox partner proteins such as cytochrome b5, a ferredoxin reductase and a ferredoxin, and adrenodoxin reductase and adrenodoxin.

Although classification and nomenclature of cytochrome P450 is quite complex, they can be classified by their redox partner transfer protein system, proposed by I. Hanukoglu (1996). "Electron Transfer Proteins of Cytochrome P450 Systems". Advances in Molecular and Cell Biology. Advances in Molecular and Cell Biology. 14: 29-56. In summary, cytochromes P450 can be classified into the following groups:

Microsomal P450 systems which utilise cytochrome P540 reductase or cytochrome b5 to transfer electrons from cofactor to cytochrome P450;

Mitochondrial P450 systems which utilise adrenodoxin reductase and adrenodoxin to transfer electrons from reduced cofactor to cytochrome P450; Bacterial P450 systems which utilise ferredoxin reductase and ferredoxin to transfer electrons from reduced cofactor to cytochrome P450;

CYB5R-cytb5-P450 systems, which utilise cytochrome b5 for the electron transfer from the cofactor to the cytochrome P450;

FMN-Fd-P450 systems in which the electron partner reductase is a fused FMN domain;

P450 only systems that do not require redox partner proteins, e.g., P450 _BM-3 ·

Isolated bacterial cytochrome P450 enzymes are known, including P450 _cam from Pseudomonas putida, J Biol Chem (1974) 249, 94; P450 _BM-I and P450 _bm-3 both from Bacillus megaterium ATCC 14581 , Biochim Biophys Acta (1985) 838, 302, and J Biol Chem (1986) 261 , 1986, 7160; P450a, P450b, and P450c from Rhizobium japonicum, Biochim Biophys Acta (1967) 147, 399; and P450npd from Nocardia NHI, Microbios (1974) 9, 119.

However, cytochrome P450 enzymes purified from Actinomycete microorganisms remain relatively unreported. The induction of a cytochrome P450 in Streptomyces griseus by soybean flour (P450 _soy ) is described in Biochem and Biophys Res Comm (1986) 141 , 405. Other reported examples include the isolation and properties of two forms of a P450 effecting pesticide inactivation (P450 _SUi & su2) and two forms of 6-deoxyerythronolide B hydroxylase from Saccharopolyspora erythraea (originally classified as Streptomyces erythraeus) as described in Biochemistry (1987) 26, 6204. US6884608 describes enzymatic hydroxylation of epothilone B to epothilone F, effected with a hydroxylation enzyme produced by a strain of Amycolatopsis orientalis (originally classified as Streptomyces orientalis).

In the field of medicinal chemistry, modifications to chemical compounds are used to modify the properties of such chemical compounds. For example, tertiary butyl moieties are often used by medicinal chemists in the synthesis of drug-like molecules for introduction of hydrophobicity. However, further modifications thereof can be used to improve potency, selectivity and solubility profiles of such compounds, for example hydroxylations can be used. Hydroxylations are also the main route of metabolic degradation, another important aspect of pharmacology and medicinal chemistry. Methods for the production of these hydroxylated metabolites are sought using biotransformation with animal tissues.

It had previously been found that a cytochrome P450 enzyme found in Amycolatopsis lurida NRRL-2430 can be used for the hydroxylation of a wide range of organic substrates.

In particular, a cytochrome P450 enzyme having the SEQ ID NO: 2 can be used for the hydroxylation of organic compounds in order to activate or modify the compound’s physicochemical and pharmacological properties. In a particularly preferred embodiment, the cytochrome P450 enzyme having the SEQ ID NO: 3 is used for the hydroxylation of isopropyl or tertiary butyl moieties, or chemicals containing such moiety, for the purposes of C-H activation or modification of the compound’s physicochemical and pharmacological properties.

Summary of the invention

It has now been found that mutagenesis to alter the sequence of this enzyme shown in SEQ ID No: 1 10 at residue T291 produces modified cytochrome P450 enzymes with improved properties including altered regiospecificity for hydroxylation of aromatic substrates. A first aspect of the invention provides the use of a cytochrome P450 enzyme comprising SEQ ID NO: 110, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, for the hydroxylation of an organic compound, wherein the amino acid residue at position 291 is not threonine.

A second aspect of the invention provides a method for the production of a hydroxylated organic compound, comprising reacting the organic compound with an enzyme preparation containing in part the cytochrome P450 enzyme comprising SEQ ID NO: 110, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, wherein the amino acid residue at position 291 is not threonine.

A third aspect of the invention provides a kit comprising i) a cytochrome P450 enzyme comprising SEQ ID NO: 110, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, or ii) a microorganism that expresses a cytochrome P450 enzyme comprising SEQ ID NO: 1 10, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, wherein the amino acid residue at position 291 is not threonine and wherein the kit further comprises instructions and other cofactor reagents for use for the hydroxylation of an organic compound.

Brief description of the figures:

Fig. 1 shows various ID sequences. SEQ ID NO: 1 is the nucleic acid sequence of P450 aluC09, ferredoxin aluF03 and ferredoxin reductase SCF15A; SEQ ID NO: 2 is the amino acid sequence of P450 AluC09; SEQ ID NO: 3 is the amino acid sequence of ferredoxin AluF03; SEQ ID NO: 4 is the amino acid sequence of ferredoxin reductase SCF15A; SEQ ID NO: 7 is the nucleic acid sequence of aluC09M1 1 ; SEQ ID NO: 8 is the amino acid sequence of AluC09M11 ; SEQ ID NO: 11 is the nucleic acid sequence of aluC09M12; SEQ ID NO: 12 is the amino acid sequence of AluC09M12; SEQ ID NO: 15 is the nucleic acid sequence of aluC09M17; SEQ ID NO: 16 is the amino acid sequence of AluC09M 17; SEQ I D NO: 19 is the nucleic acid of aluC09M 18; SEQ ID NO: 20 is the amino acid sequence of AluC09M18; SEQ ID NO: 23 is the nucleic acid sequence of aluC09M19; SEQ ID NO: 24 is the amino acid sequence of AluC09M19; SEQ ID NO: 27 is the nucleic acid sequence of aluC09M20; SEQ ID NO: 28 is the amino acid sequence of AluC09M20; SEQ ID NO: 31 is the nucleic acid sequence of aluC09M21 ; SEQ ID NO: 32 is the amino acid sequence of AluC09M21 ; SEQ ID NO: 35 is the nucleic acid sequence of aluC09M31 ; SEQ ID NO: 36 is the amino acid sequence of AluC09M31 ; SEQ ID NO: 39 is the nucleic acid sequence of aluC09M32; SEQ ID NO: 40 is the amino acid sequence of AluC09M32; SEQ ID NO: 43 is the nucleic acid sequence of aluC09M33; SEQ ID NO: 44 is the amino acid sequence of AluC09M33; SEQ ID NO: 47 is the nucleic acid sequence of aluC09M34; SEQ ID NO: 48 is the amino acid sequence of AluC09M34; SEQ ID NO: 51 is the nucleic acid sequence of aluC09M35; SEQ ID NO: 52 is the amino acid sequence of AluC09M35; SEQ ID NO: 55 is the nucleic acid sequence of aluC09M36; SEQ ID NO: 56 is the amino acid sequence of AluC09M36; SEQ ID NO: 59 is the nucleic acid sequence of aluC09M37; SEQ ID NO: 60 is the amino acid sequence of AluC09M37; SEQ ID NO: 63 is the nucleic acid sequence of aluC09M38; SEQ ID NO: 64 is the amino acid sequence of AluC09M38; SEQ ID NO: 67 is the nucleic acid sequence of aluC09M39; SEQ ID NO: 68 is the amino acid sequence of AluC09M39; SEQ ID NO: 71 is the nucleic acid sequence of aluC09M40; SEQ ID NO: 72 is the amino acid sequence of AluC09M40; SEQ ID NO: 75 is the nucleic acid sequence of aluC09M22; SEQ ID NO: 76 is the amino acid sequence of AluC09M22; SEQ ID NO: 79 is the nucleic acid sequence of aluC09M23; SEQ ID NO: 80 is the amino acid sequence of AluC09M23; SEQ ID NO: 83 is the nucleic acid sequence of aluC09M24; SEQ ID NO: 84 is the amino acid sequence of AluC09M24; SEQ ID NO: 87 is the nucleic acid sequence of aluC09M25; SEQ ID NO: 88 is the amino acid sequence of AluC09M25; SEQ ID NO: 91 is the nucleic acid sequence of aluC09M26; SEQ ID NO: 92 is the amino acid sequence of AluC09M26; SEQ ID NO: 95 is the nucleic acid sequence of aluC09M27; SEQ ID NO: 96 is the amino acid sequence of AluC09M27; SEQ ID NO: 99 is the nucleic acid sequence of aluC09M28; SEQ ID NO: 100 is the amino acid sequence of AluC09M28; SEQ ID NO: 103 is the nucleic acid sequence of aluC09M29; SEQ ID NO: 104 is the amino acid sequence of AluC09M29; SEQ ID NO: 107 is the nucleic acid sequence of aluC09M30; SEQ ID NO: 108 is the amino acid sequence of AluC09M30; SEQ ID NO: 109 shows the nucleic acid sequence where the nucleotides at positions 141 to 143, 223 to 225, 871 to 873 and 943 to 945 are denoted as modified; SEQ ID NO: 110 shows the amino acid sequence of the enzyme of the invention, where the residue at position 47, 75, 291 and 315 are denoted as modified.

Fig. 2 shows carbon monoxide difference spectra for a specific AluC09 mutant as described in Example 4.

Fig. 3 shows example UPLC chromatograms monitoring the hydroxylation of diclofenac catalysed by AluC09 and various T291x mutants.

Fig. 4 shows UPLC chromatograms with UV detection at 275 nm showing the hydroxylation of flufenamic acid catalysed by, from top to bottom: cytochrome P450 aluC09 and mutants aluC09M18, aluC09M35 and aluC09M39.

Fig. 5 shows UPLC chromatograms with UV detection at 275 nm for the hydroxylation of aceclofenac catalysed by various enzymes of the invention.

Fig. 6 shows UPLC chromatograms with UV detection at 300 nm for the metabolism of napropamide catalysed by various enzymes of the invention.

Fig. 7 shows UPLC chromatograms showing O-demethylation of indomethacin catalysed by P450 mutant aluC09M36.

Fig. 8 shows the structural formulae of selected compounds utilised in Example 5.

Description of the preferred embodiments

A first aspect of the invention provides the use of a cytochrome P450 enzyme comprising SEC ID NO: 110, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, for the hydroxylation of an organic compound, wherein the amino acid residue at position 291 is not threonine.

Specifically, the present invention provides the use of the enzyme cytochrome P450 _aiu co ₉ having a mutation at position 291. This enzyme has amino acid sequence as shown in SEC ID NO: 1 10.

The parent enzyme is present in the strain Amycolatopsis lurida, deposited in the ARS Culture Collection, National Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois 61604, USA, under the Accession number NRRL 2430. The strain has also been deposited with the American Tissue Culture Collection, with the accession number ATCC 14930.

When the specific mutation of this enzyme, or a variant thereof, is combined with suitable reductase components, it is able to hydroxylate organic compounds.

The enzyme cytochrome P450 _aiu co ₉ can be extracted, with or without purification from the known Amycolatopsis lurida NRRL-2430, or other bacterial strain, or similarly extracted, with or without purification from a recombinant expression system via cloning of cytochrome P450 _aiu co ₉ into an expression system, such as E.coli, as will be understood by the skilled person.

Actinomycetes including Amycolatopsis lurida NRRL-2430 readily undergo mutation both through natural causes and as a result of artificial treatments such as UV irradiation, radiation treatment and chemical treatment. The present invention embraces all productive mutants of Amycolatopsis lurida NRRL-2430. These mutant strains also include any strains obtained by gene manipulation such as gene recombination, transduction and transformation. It is also well-known that the properties of Actinomycetes change in some degree even for the same strain after successive cultures. Therefore, strains cannot always be differentiated taxonomically because of a slight difference in culture properties. This invention embraces all strains that can produce one or more of the cytochromes P450 enzymes, and especially strains that cannot be clearly differentiated from strain NRRL-2430 or its mutants.

One of skill in the art will appreciate that the present invention can include variants of those particular amino acids sequences which are exemplified herein. Particularly preferred are variants having an amino acid sequence similar to that of the amino acid sequences disclosed herein, in which one or more amino acids residues are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein of the present invention. Various amino acids have similar properties, and one or more such amino acids of a substance can often be substituted by one or more other amino acids without eliminating a desired activity of that substance. Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Variants include naturally occurring and artificial variants. Artificial variants may be generated using mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms. Preferably, the variants have substantial identity to the amino acid sequences exemplified herein. As used herein, the term “variant” or “mutant thereof refers to amino acid sequences which have "substantial identity", preferably having at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 98.1 %, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.1 %, 99.8% or 99.9% identity with SEQ ID NO 1 10. Desirably, the term "substantial identity" indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art amino acid sequences. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. The above applies mutatis mutandis to all amino acid sequences disclosed in the present application.

“In a preferred embodiment, the term “variant” or “mutant thereof generally refers to a sequence having at least 70% identity to SEQ ID No 1 10 and CYP450 activity, more preferably least 90% identity thereto or at least 95% identity thereto, further preferably 96% identity thereto, even more preferably 97% identity thereto, most preferably 100% identity thereto.

With regard to“variants” or“mutants”, it will be understood that the amino acid at position 291 is not threonine. The mutation at residue position 291 (shown in SEQ ID NO: 110) may be any amino acid other than threonine. In preferred embodiments, the threonine residue in the parent sequence has been replaced with the amino acid glutamic acid (E), glutamine (Q), alanine (A) asparagine (N), aspartic acid (D), tyrosine (Y) or valine (V). Most preferably, the threonine is replaced with glutamic acid (E).

The invention also envisages one or more additional mutations being incorporated into the enzyme sequence. Additions, substitutions and deletions can all be made, as will be appreciated by the skilled person. Preferably, the enzyme may incorporate a replacement amino acid at one or more of positions A47, M75 and V315, based on the sequence shown as SEQ ID NO: 1 10.

A variety of different compounds can be hydroxylated using the claimed cytochrome P450 enzyme. In a preferred embodiment, the organic compound to be hydroxylated will have a rate of conversion to the hydroxylated derivative of at least 3%, more preferably at least 5%, more preferably at least 10%, more preferably at least 25%, more preferably at least 50%, even more preferably at least 70% and most preferably a rate of conversion to the hydroxylated derivative of 100%, using the same conditions described in Example 4 herein.

Preferably, the organic compound to be hydroxylated is not epothilone.

The compound to be hydroxylated by the cytochrome P450 enzyme may have an optionally substituted branched alkyl group, such as isopropyl or tert butyl, which is hydroxylated; or an aromatic group, such as an optionally substituted aryl or heteroaryl, which is hydroxylated.

There is a particularly high conversion rate from these compounds to their hydroxylated derivatives when using the claimed cytochrome P450 enzyme.

Preferably, the compound to be hydroxylated is of formula la or lb:

R R

R ^“ R ^' (la) (lb)

where R represents the rest of the compound, and where R ¹ , R ² and R ³ are independently selected from H or Ci_i ₂ alkyl or C _6-i o aryl, or wherein any two of R ¹ , R ² and R ³ may be joined to form an optionally substituted cycloalkyl or heterocycloalkyl or R ¹ , R ² and R ³ may be joined together with their bridging carbon to form an olefin, aryl or heteroaryl, and wherein A is optionally substituted benzyl, aryl or heteroaryl.

Preferably R is an optionally substituted alkyl; an optionally substituted olefin, an optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocycloalkyl.

Preferably, A is benzyl, aryl or heteroaryl, optionally substituted with one or more halogen atoms, Ci-C ₈ alkyl, CrC ₈ alkoxy, -COOH, C _I -C ₈ -COOH, cyano, amino or alkylamino groups.

More preferably, A is benzyl, aryl or heteroaryl optionally subsitituted with one or more halgen atoms, Ci-C ₄ alkyl, -COOH or Ci-C ₄ Alkyl-COOH groups.

More preferably, A is C ₆ aryl or C ₄ -C ₂₀ bicyclic or tricyclic heteroaryl, optionally substituted with Ci-C ₈ alkyl, C _r C ₈ alkoxy, -COOH, C _r C ₈ -COOH, cyano, amino or alkylamino groups.

Most preferably, A is indolyl, isindolyl, azaindolyl or a group having the formula.

As used herein“alkyl” means a C1-C10 alkyl group, which can be linear or branched or cyclic. Examples include propyl and butyl, pentyl, hexyl, cyclopentyl and cyclohexyl. Preferably, it is a C ₃ -Ci ₀ alkyl moiety. More preferably it is a C ₅ - C ₆ alkyl moiety. Preferably the alkyl is an optionally substituted cyclohexyl.

As used herein“alkoxy” means on alkyl group, as defined above, having an oxygen atom attached thereto.

As used herein“halogen” refers to fluorine, chlorine, bromine and iodine.

As used here the term“alkylamino” means at least one alkyl group, as defined herein, is appended to the parent molecular moiety though an amino group. By way of non-limiting example, suitable alkylamino groups include methlamino, ethlamino, proylamino, butylamino and hexylamino.

The term “alkenyl” means a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond and from 1 to 10 carbon atoms.

For the avoidance of any doubt, the term cycloalkyl is a cyclic alkyl group.

As used herein“aryl” means an optionally substituted monocyclic, bicyclic or tricyclic aromatic radical, such as phenyl, biphenyl, napthyl, anthracenyl. Preferably the aryl is an optionally substituted C ₆ aryl.

As used herein“heteroaryl” means an optionally substituted monocyclic, bicyclic or tricyclic aromatic radical containing at least one and up to four heteroatoms selected from oxygen, nitrogen and sulfur, such as furanyl, pyrrolyl, thiazolyl, isothiazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl, thienyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, indolyl, azaindolyl, isoindolyl, quinolyl, isoquinolyl, triazolyl, thiadiazolyl, oxadiazolyl. Preferably the heteroaryl is an optionally substituted thioazole.

As used herein “heterocycloalkyl” means an optionally substituted cycloalkyl wherein one to four carbon atoms have been substituted with a heteroatom. Preferably, the heteroatoms are selected from nitrogen, oxygen, sulphur or phosphorous.

As used herein the term“optionally substituted” means an H has been removed from a compound and replaced with an organic fragment such as those those comprising a combination of any of carbon, hydrogen, nitrogen, oxygen and sulphur. Preferably the compound of formula I has a molecular weight of from 50 to 2000, such as from 100 to 700, more preferably from 200 to 500.

Preferably at least 2 of R ¹ , R ² and R ³ are selected from Ci-i ₂ alkyl or C _6-i o aryl. Preferably, R ¹ , R ² and R ³ are independently selected from H, Ci _-6 alkyl or C _6-i o aryl, preferably with the proviso that either one or none of R ¹ , R ² and R ³ is H. Most preferably, R ¹ , R ² and R ³ are independently selected from H, methyl, ethyl, propyl, butyl, t-butyl, pentyl and hexyl preferably with the proviso that either one or none of R ¹ , R ² and R ³ is H.

In a particularly preferred embodiment, the compound to be hydroxylated is of formula (II), where R represents the rest of the compound and where R ¹ is CHs or H:

R

)T ^r1

H ₃ C CH ₃ (II)

In this case, the cytochrome P450 enzyme catalyses the following reaction:

Where R ¹ = H, CH ₃

Therefore, in a particular preferred embodiment, the organic compound contains an isopropyl or tert-butyl group. Most preferably, the group to be hydroxylated is a tert-butyl moiety. The cytochrome P450 enzyme catalyses the conversion of a substrate compound with a tert-butyl moiety to a hydroxylated- tert-butyl derivative.

In a particularly preferred embodiment, the cytochrome P450 enzyme is reacted with a compound such as bosentan, diclofenac, buparvaquone, tivantinib, BIRB796 or ritonavir. Most preferably, the cytochrome P450 enzyme is reacted with bosentan, buparvaquone, BIRB796 or ritonavir. The compounds of formula I are typically of the following structural formulae:

The mutated cytochrome P450 enzyme, or variants thereof, wherein the amino acid residue at position 291 is not threonine, may optionally be used in combination with reductase components, which activate the cytochrome P450. In a preferred embodiment, ferredoxin and ferredoxin reductase components are used. Any components which activate the cytochrome P450 may also be used, including small-molecule chemicals acting directly or indirectly, protein chemicals in solution or those fused directly or by peptide linkage, or electronic via electrode. In a particularly preferred embodiment, the enzyme cytochrome P450aluC09 having SEQ ID NO 1 10, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, is combined with suitable ferredoxin and ferredoxin reductase components to give an effective system to convert a substrate compound to a hydroxylated derivative.

In a preferred embodiment, parent the cytochrome P450 enzyme or variant thereof is present in Amycolatopsis lurida (NRRL-2430) cells.

In another preferred embodiment, the cytochrome P450 enzyme or variant thereof is expressed by at least one recombinant microorganism comprising heterologous nucleic acid encoding the enzyme, derived from Amycolatopsis lurida (NRRL 2430). As used herein the term“comprising” is intended to mean containing at least the claimed sequence, but may include other sequences. In one embodiment, the recombinant microorganism comprises a heterologous nucleic acid encoding the enzyme or variant thereof. In an alternative embodiment, the recombinant microorganism also comprises a heterologous nucleic acid encoding a reductase agent.

In another aspect of the invention, there is provided a method for the production of a hydroxylated organic compound, comprising reacting the organic compound with a cytochrome P450 enzyme comprising SEQ ID NO: 1 10, or a variant enzyme having at least 70% identity thereto and having CYP450 activity.

The choice of compound to be hydroxylated is discussed above.

In a preferred embodiment, the enzyme is used to catalyse the hydroxylation of a propyl group or a butyl group, more preferably an isopropyl or isobutyl group or tert-butyl group. Most preferably, the enzyme is used to catalyse the hydroxylation of a tert-butyl moiety. The cytochrome P450 enzyme is able to catalyse the conversion of a substrate compound with a tert-butyl moiety to a hydroxylated- tert-butyl derivative.

In a particularly preferred embodiment, the compound to be hydroxylated is bosentan, diclofenac, tivantinib, buparvaquone, BIRB796 or ritonavir. Most preferably, the compound to be hydroxylated is bosentan, buparvaquone, BIRB796 or ritonavir.

Optionally, one or more additional component(s) may be used to activate the cytochrome P450 enzyme. In an embodiment according to the present invention, the cytochrome P450 enzyme is used in combination with reductase components, preferably with ferredoxin and ferredoxin reductase components.

The cells comprising the enzyme of the invention may be dosed with the organic compound to be hydroxylated. The method may optionally comprise an additional step wherein the cells are subsequently harvested and purified to obtain the hydroxylated compound.

Culture of the recombinant cells expressing the enzyme of the invention is suitably performed by seeding of a conventional culture medium containing nutrients well-known for use with such microorganisms. Thus, the culture medium contains sources of assimilable carbon and of assimilable nitrogen. The culture medium may also contain inorganic salts. Examples of sources of assimilable carbon include glucose, sucrose, starch, glycerin, millet jelly, molasses and soybean oil. Examples of sources of assimilable nitrogen include soybean solids (such as soybean meal or soybean flour), wheat germ, meat extracts, peptone, corn steep liquor, dried yeast and ammonium salts, such as ammonium sulphate. If required, inorganic salts, such as sodium chloride, potassium chloride, calcium carbonate and various phosphates, may also be included. The medium is preferably sterilized and has a pH adjusted to 5 to 8.

The skilled person will understand that the particular cultivation technique employed is not critical to the invention and any technique commonly used for the cultivation of the specific recombinant bacteria may equally be employed with the present invention. For example, the bacteria may be an Actinomycete, and suitable culture conditions will be apparent to the skilled person. In general the techniques employed will be chosen having regard to industrial efficiency. Thus, liquid culture is generally preferred and the submerged culture method is most convenient from the industrial point of view. Cultivation is preferably carried out under aerobic conditions.

The enzymes of this invention are inducible enzymes, and are not produced unless an induction agent is present. For preference, but not limited to, the induction agent is selected to be the same as the intended substrate for the isolated enzyme. When from 4 hours to 3 days have elapsed after inoculation, preferably 0.05 to 5 mM, more preferably 0.2 mM of induction agent is added, and then cultivation is continued for 2 hours to 1 week, preferably for about one day. The temperature of cultivation is typically 20° to 45° C., preferably 25° to 30° C., optimally about 27° C. Shake culture or aeration techniques can be adopted.

The cells obtained by the cultivation may be disrupted by cell disruption techniques such as high-pressure homogenisation in buffer solution. The supernatant obtained by centrifugation gives the crude enzyme solution. For example, the enzyme of the present invention can be obtained in a supernatant produced by centrifugation at 38,000xg for 20 minutes.

In an alternative embodiment, the cytochrome P450 enzyme or variant thereof wherein the amino acid residue at position 291 is not threonine is expressed by at least one recombinant microorganism comprising heterologous nucleic acid encoding the enzyme, derived from Amycolatopsis lurida (NRRL 2430). Here, the at least one recombinant microorganism can be dosed with an organic compound to be hydroxylated. This method may optionally comprise a purification step to obtain the hydroxylated compound.

In a preferred embodiment, this can be achieved by the recombinant expression of the functional cytochrome P450aluC09 protein with intact haem. This can be expressed with any or all of the cofactor enzymes. In a particularly preferred embodiment, ferredoxin and ferredoxin reductase may be expressed. This can be achieved by polycistronic plasmid use or via fusion-protein, either via linkers or directly into a single protein product.

Alternatively, the functional mutant cytochrome P450aluC09 protein, or variant thereof, wherein the amino acid residue at position 291 is not threonine, may be expressed alone without mixing with cofactor enzymes. In a preferred embodiment, cofactor enzymes may be titrated in to provide the active enzyme reaction after material production. The cofactors may be obtained by extraction from wild-type or recombinant materials derived from plants or microbial fermentation. Hussain & Ward,. Appl Environ Microbiol. 2003; 69(1 ):373-382, describe the cloning techniques that may be used.

The native organism, host strain expressing the recombinant enzyme or extracted enzyme is contacted directly with the substrate, preferably in an aqueous medium, either mono, bi or triphasic, with such multiphase systems being either in dispersed or layered form. Reaction conditions, including choice of pH and temperature will be evident to the skilled person, based on conventional techniques. For example, a selected microbial growth medium or phosphate buffer solution at a pH value in the range of from 5 to 12, more preferably 6.5 to 11.0, most preferably around 7.4 may be used. Of particular note is the activity of this recombinant enzyme at elevated pH values e.g., pH value of 11. The ability to catalyse reactions at such a pH affords a particular commercial advantage because increased substrate loading may be achieved for selected substrates with improved solubility at a higher pH, such as compounds with a carboxyl moiety. Other advantages of catalysis at higher pH are the ability to directly utilise the product from a prior step resulting in such products, such as chemical synthesis, base-catalysed hydrolysis of a feedstock, or reaction product from another enzyme where increasing pH has been used to stop that reaction. The reaction temperature is preferably within the range from 10° to 45° C., more preferably from 25° to 30° C. The concentration of the substrate in the reaction medium is preferably within the range from 0.01 to 5.0% by weight. The time allowed for the reaction is normally from 1 minute to 5 days, more usually from 1 hour to 16 hours, although this may vary, depending upon the concentration of substrate in the reaction mixture, the reaction temperature, and other factors. The extracted enzyme material can either be used directly after extraction, after storage in frozen solution. In a particularly preferred embodiment, the extracted enzyme material can be dried, preferably by lyophilisation, for later use with or without the addition of other components required for reaction, such as other enzyme cofactor components.

After completion of the conversion reaction, the hydroxylated compound can be isolated using conventional procedures, including, for instance, filtration, solvent extraction, chromatography, crystallization, and other isolation procedures. Such procedures will be selected having due regard to the identity of the product. Before, during or after the isolation, the product may or may not be derivatised, as desired.

The starting materials as substrates for the enzyme may by either derived from synthetic routes, naturally occurring, either via natural biomass such as plant material, or produced by fermentation, or by mixed routes thereof. Enzyme reactions can also be performed using pure or non-purified materials, the resulting reaction may be used to aid later purifications of reacted or unreacted components.

Of the substrate compounds used as starting materials, free bases, alkali metal salts, e.g. the sodium or potassium salts, or acid salts of organic or inorganic nature such as tosylate or hydrochlorides, are suitable for use.

After completion of the conversion reaction, the desired compound can be obtained from the reaction system, collected, isolated and purified by conventional means if required, or onward used directly in unpurified form. For example, the reaction product is centrifuged or filtered and the supernatant or filtrate is extracted with a hydrophobic resin, ion-exchange resin or water- immiscible organic solvent such as ethyl acetate. After evaporation of the solvent of the extract, the remaining crude hydroxylated compound, for example the remaining crude hydroxylated tert-butyl compound, may be purified by subjecting it to column chromatography using silica gel or alumina or reversed-phase stationary phase, and by eluting with a suitable eluent. If the starting material is a mixture, then the product can be isolated as a mixture of hydroxylated compounds which if desired can be separated using chromatography or other suitable techniques.

In general, the hydroxylated compounds may have improved pharmaceutical or agrochemical properties, such as bioactivity potency, improved solubility characteristics, reduced off-target interactions, or simply of further utility, such as for onward synthesis, or be useful for an analytical standard. Particularly preferred are the hydroxylated compounds of formulas (I) & (II) discussed above.

The present invention is further illustrated with reference to two classes of substrates of markedly different structure, namely aromatic-tBu compounds such as bosentan, and compounds in which the tBu is not directly bonded to an aromatic carbo-cycle system such as buparvaquone.

When the cytochrome P450 enzyme preparation of this invention is reacted with diclofenac as substrate at pH 7.4 for 5 minutes with (a) ferredoxin, (b) ferredoxin-NADP ⁺ -reductase, (c) NADP ⁺ , (d) NADPH regeneration system, and (e) dissolved oxygen, the temperature of reaction ranges at least from 4° C. to 60° C. The optimum pH for each cytochrome ranges from 6.5 to 1 1.0. Each cytochrome is stable when kept for 24 hours at 4° C at pH 7.4.

The use of ferredoxin, ferredoxin-NADP ⁺ -reductase, oxygen and NADPH is not essential. Any components which can activate the cytochrome P450 may be adopted.

Measurement of the enzyme activity is normally effected in one of two ways:

(i) Measurement on cytochrome P450 _aiu co9 Measurement is performed according to the method of Omura and Sato et al. (J Biol Chem, 239. 1964, 2370). That is to say, cytochrome P450 _aiu co ₉ is analyzed quantitatively using the following formula, based on the difference in the absorbance of the reduced CO versus the reduced difference spectrum at 450 nm and 490 nm.

Abs(4S0nm Abs(490nm)

Cytochrome P 450 (mM) - - — ——

91 (mM cm x l (cm)

(ii) Measurement of rate of formation of hydroxylated diclofenac derivatives from diclofenac

The following cocktail of components is employed:

Potassium phosphate buffer pH 7.4 50 mM

MgCI ₂ 5 mM

Enzyme solution containing expressed Fd, FdR, P450 native

concentration as extracted

NADP ⁺ 1 mM

Glucose-6-phosphate 5 mM

Glucose-6-phosphate dehydrogenase 2 UN/ml

Diclofenac substrate 0.125 mg/ml

Total volume 0.50 ml

To measure enzyme activity the components of the table are mixed, the solution is shaken at 30° C for 16-20hours, and then 500 pi of acetonitrile is added and the reaction stopped. The amount of hydroxy-diclofenac formed by the enzyme system is determined with HPLC or UPLC.

Using the test methods for determining activity, the loss of activity with change in temperature and pH can be determined.

For example, the cytochrome is fully inactivated at pH 7.4 and 70° C. for 60 minutes in the presence of 20% glycerol and 2 mM dithiothreitol. The cytochrome is inactivated at pH 3 or a more acidic pH. In a further aspect, the invention provides a kit comprising i) a cytochrome P450 enzyme comprising SEQ ID NO: 1 10, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, or ii) a microorganism that expresses a cytochrome P450 enzyme comprising SEQ ID NO: 1 10, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, wherein the amino acid residue at position 291 is not threonine and wherein the kit further comprises instructions for use for the hydroxylation of an organic compound.

The kit allows the user to screen for the hydroxylation of compounds of interest.

In a preferred embodiment, the kit further comprises electron donating agents. The kit may preferably comprise as the electron donating agents ferredoxin reductase and a ferredoxin with cofactors NADH or NADPH or cofactor regeneration systems such as NAD+ or NADP+, glucose or glucose-6- phosphate, and glucose-dehydrogenase or glucose-6-phosphate dehydrogenase. However, any suitable electron donating agents may be used.

Optionally, the kit may further comprise a buffer, either separately or contained with the other components.

Preferably, the kit may further comprise one or more other CYP450 enzymes.

Preferably, the cytochrome P450 enzyme or microorganism is lyophilised or immobilised or tethered to other macromolecules or support materials such alginate beads, Nickel columns and electrochemical electrodes.

The methods of the present invention are demonstrated in the examples below. These examples are provided as an illustration only and should not be construed as limiting on the present invention.

Examples

Example 1 : Cloning of P450 _aiu co ₉ T291 Mutants

PCR-based Site Directed Mutagenesis

Site directed mutagenesis by PCR of position T291 in P450 _aiu co ₉ was performed in which the individual threonine codon was altered to a new amino acid. Seven new amino acids were chosen to modify position 291. The sequences of primers used for amplification and mutagenesis are shown in Table 1. Two 23 mer oligonucleotides were designed that are complementary to the sequences surrounding the ACG triplet encoding the threonine residue.

Table 1

This method used pQR368bb-aluC09-aluF03 as the template DNA and mutagenic primers. PCR reactions contained 12.5 pi of Phusion ^® High-Fidelity PCR Master Mix (1 U/mI; New England Biolabs), ~ 2 ng of template DNA, 1.25 pi DMSO, 0.5 mM of each forward and reverse primer and the reaction was filled up to a total volume of 25 mI with MilliQ ^® -H ₂ 0. Since the whole of the plasmid was amplified leading to long PCR products, reactions were supplemented with 5% DMSO. Amplification reactions were identical for all mutagenic reactions, with the exception of annealing temperatures which varied as follows: 58 °C for mutants 1 1 , 12, 61 °C for mutant 17, 62 °C for mutants 18, 20, 64 °C for mutant 19, and 60 °C for mutant 21. Reactions were performed on a Techne™ TC-512 Thermal Cycler with the following cycling conditions: 98 °C for 30 seconds, 16 cycles (98 °C for 30 seconds, annealing temperature for 1 minute, 72 °C for 8 minutes), 72 °C for 10 minutes. Amplifications were then subjected to Dpnl digestion.

Dpnl digestion

One microliter of Dpnl (20 U/mI; New England Biolabs), 1 mI Cutsmart buffer (New England Biolabs) was added to 8 mI of the PCR reaction. Unmutated template DNA was digested for 60 min at 37 °C.

Cloning of mutants

Dpnl reactions were used to transform 50 mI chemically competent E. coli DH5a cells. Clones were selected on lysogeny broth (LB) plates containing 100 pg/ml ampicillin after 16 hours of incubation at 37 °C. Clones were picked and cultivated in 5 ml LB containing 100 pg/ml ampicillin for 16 hours at 37 °C and 250 rpm. Recombinant plasmids were isolated from these cultures using the QIAprep ^® Spin Miniprep Kit (Qiagen) and analysed via DNA sequencing.

DNA sequencing and analysis

DNA sequences of the cloned mutants and the reductase part of the pQR368bb vector backbone were confirmed by Sanger sequencing at Eurofins Genomics (Germany).

Example 2: Cloning of P450 _aiuC o ₉ Double Point Mutants

PCR-based Site Directed Mutagenesis Site directed mutagenesis by PCR of position T291 in P450 _aiu co ₉ was performed in which the individual threonine codon was altered to a new amino acid. This created Mutant 12 (T291 E) as described in Example 1. This mutant was then used as the template DNA for further site directed mutagenesis of positions A47, M75 and V315, thus producing the double point mutants 22-30. The sequences of primers used for amplification and mutagenesis are shown in Table 2. Two 23 mer oligonucleotides were designed that are complementary to the sequences surrounding the ACG triplet encoding the threonine residue.

This method used P450 _aiu co ₉ T291 E (Mutant 12) as the template DNA and mutagenic primers. PCR reactions contained 12.5 pi of Phusion ^® High-Fidelity PCR Master Mix (1 U/mI; New England Biolabs), ~ 2 ng of template DNA, 1.25 mI DMSO, 0.5 mM of each forward and reverse primer and the reaction was filled up to a total volume of 25 mI with MilliQ ^® -H ₂ 0. Since the whole of the plasmid was amplified leading to long PCR products, reactions were supplemented with 5% DMSO. Amplification reactions were identical for all mutagenic reactions, with the exception of annealing temperatures which varied as follows: 52 °C for mutants 23, 24, 64 °C for mutants 22, 25, 28, 30, 66 °C for mutant 29, and 69 °C for mutants 26, 27. Reactions were performed on a Techne™ TC-512 Thermal Cycler with the following cycling conditions: 98 °C for 30 seconds, 16 cycles (98 °C for 30 seconds, annealing temperature for 1 minute, 72 °C for 8 minutes), 72 °C for 10 minutes. Amplifications were then subjected to the same Dpnl digestion, cloning of mutants and DNA sequencing and analysis as in Example 1.

Table 2

Example 3: Expression of mutants

Construction of the recombinant expression strain

The strain E. coli BL21 (DE3) (Invitrogen) was used as a host for recombinant expression of mutant variants of P450 _aiu co ₉ , ferredoxin _aiUF03 and ferredoxin reductase _{SCFi 5A} · To construct this expression strain, E. coli BL21 (DE3) cells were transformed with plasmid using the heat shock procedure. Twenty five microliters of chemically competent cells were mixed with 1 pi (-100 ng) of mutant plasmid followed by incubation on ice for 30 minutes . Heat shock was performed for 30 seconds in a water bath at 42 °C and cells were subsequently chilled on ice for 2 min. After the addition of 950 pi LB Miller Media (Sigma), cells were incubated for 1 hour at 37 °C and 250 rpm in a New Brunswick Scientific Innova 4230. Two hundred microliters of the transformation mixture was plated on LB plates containing 100 pg/ml ampicillin. Plates were incubated at 37 °C for approximately 16 hours.

Expression of recombinant mutant AluC09, AluF03 and SCF15A

To prepare glycerol stocks of this expression strain, a streak of colonies were picked and inoculated into 5 ml LB containing 100 pg/ml ampicillin and cultivated at 37 °C and 250 rpm for approximately 16 hours. Five hundred microliter of this culture were mixed with 500 mI of 50% glycerol (weighhvol) in cryovials and stored at -80 °C.

Preculture: Five milliliters of LB Miller media supplemented with 100 pg/ml of ampicillin was inoculated with a loop scraped from a cryovial containing of E.coli BL21 (DE3) harbouring the mutant plasmid. Cells were grown overnight at 37°C and 250 rpm in a New Brunswick Scientific Innova 4230.

Seed: Into a 250 ml baffled flask, 50 ml of PCM8.1 media supplemented with 100 pg/ml of ampicillin was inoculated with the overnight preculture to an OD600 of 0.1 and incubated at 37°C and 200 rpm until the end of the day.

The components of PCM8.1 were MgS0 ₄ (0.49 gl_ ¹ ), Na ₂ HP0 _4* 7H ₂ 0 (6.7 gl_ ¹ ), KH ₂ P0 ₄ (3.4 gL ¹ ), NH ₄ CI (2.68 gL ¹ ), Na ₂ S0 ₄ (0.71 gL ¹ ), arginine (0.2 gL ¹ ), histidine (0.15 gL ¹ ), lysine (0.2 gL ¹ ), phenylalanine (0.2 gL ¹ ), serine (0.2 gL ¹ ), threonine (0.2 gL ¹ ), tryptophan (0.2 gL ¹ ), methionine (0.2 gL ¹ ), monosodium glutamate (8 gL ¹ ), glucose (0.5 gL ¹ ), glycerol (10 gL ¹ ) and a 1000-fold diluted trace element solution with FeCI3 (81.1 gL ¹ ), CaCI _2* 6H ₂ 0 (4.38 gL ¹ ), MnCI _2* 4H ₂ 0 (1.98 gL ¹ ), ZnS0 _4* 7H ₂ 0 (2.88 gL ¹ ), CoCI _2* 6H ₂ 0 (0.48 gL ¹ ), CuCI _2* 2H ₂ 0 (0.34 gL ¹ ), NiCI _2* 6H ₂ 0 (0.48 gL ¹ ), Na ₂ Mo0 _4* 2H ₂ 0 (0.48 gL ¹ )., Na ₂ Se0 ₃ (0.35 gL ¹ ), and H ₃ B0 ₃ (0.12 gL ¹ ).

Production: At the end of the day, a 1 L baffled flask containing 200 mL of PCM8.1 media supplemented with 100 pg/ml of ampicillin, 23.8 pg/ml of IPTG, 320 pg/ml of 5‘-aminolevulinic acid and 55 pg/ml of FeS0 _4* 7H ₂ 0 were inoculated with the seed cultures to an OD of 0.6. The induced production cultures were incubated at 27°C and 200 rpm until the cultures had reached stationary phase (approximately 16-20 hours). The cultures were harvested by centrifugation at 3,000 rpm for 15 minutes. The pellets were washed with 30 mL of wash buffer (isotonic 0.85% NaCI with 5% glycerol) and transferred into a fresh 50 mL falcon tube. The cells were further centrifuged at 4,000 rpm for 25- 35 minutes and the pellet was stored at -20°C for processing.

Example 4: Extraction & processing of enzyme materials

Suspended cell pellets were provided as described in Example 3, containing recombinant mutants of CYP450 _aiu co ₉ , ferredoxin _aiUF 03 and ferredoxin reductase _S c _Fi 5 _A in 50mM potassium phosphate buffer pH 7.4, 5 mM MgCI ₂ , 0.1 mM TCEP, and 1 mM PMSF in a ratio of 15 ml of buffer per 1 g of cells. Lysed cells were produced by high pressure disruption using three cycles of 30 kpsi. Lysed material was centifuged at 38,000xg for 40 minutes (4°C) and the supernatant was sterilized by passing through 0.22micron filter to provide the enzyme preparation containing the recombinant mutant CYP450 _aiu co ₉ The crude extract was then dispensed into glass vials (0.5ml per 2ml vial), frozen and lyophilised using an Edwards Supermodulyo Freeze-dryer before being stored in a standard laboratory freezer at -20°C until required for use.

Example 5: Biocatalytic activity/spectrum testing

Lyophilised material of mutants of CYP450aluC09, ferredoxinaluF03 and ferredoxin reductaseSCF15A was made as described in examples 3 and 4 and suspended in water (0.5 mL per vial) and biocatalysis was performed at 27°C in the following conditions: 50 mM potassium phosphate pH 7.4, 5 mM MgCI2, 0.125 mg/ml substrate compound such as diclofenac (Sigma-Aldrich, UK), Aceclofenac (Tokyo Chemical Industry UK Ltd, UK), Indomethacin (Sigma- Aldrich, UK), Flufenamic acid (Sigma-Aldrich, UK), Napropamide (Sigma-Aldrich, UK), celecoxib (LC Laboratories, USA), valsartan (Tokyo Chemical Industry UK Ltd, UK), ruxolitinib (LC Laboratories, USA), 4-pyrrolidylacetophenone (Sigma- Aldrich, UK), tofacitinib (LC Laboratories, USA), bosentan (CarboSynth Ltd, UK), buparvaquone (MedChemtronica, Sweden), BIRB796 (Stratech Scientific Limited, UK), epothilone B (LC Laboratories, USA), ritonavir (Tokyo Chemical Industry UK Ltd, UK) or tivantinib (MedChemtronica, Sweden), P450AluC09 (concentration as in Table 8), 5 mM G6P, 1 mM NADP, 2 UN/ml G6PDH in a final volume of 100 pL. After 16-20 hours at 200 rpm (5cm orbital shaker, Kuhner AG, Switzerland), reactions were extracted with an equal volume of acetonitrile, centrifuged to remove precipitated proteins and conversion assessed by UPLC- MS analysis.

UPLC data was obtained as follows:

Column: Acquity UPLC BEH Shield RP18 1.7pm 2.1 mm i.d. 50mm length

Solvents: H20, B: Acetonitrile, both with 0.1 % Formic acid

Flow rate: 1.0ml/min

Detector: Waters Acquity UPLC PDA (UV-Vis detection) and Waters Acquity UPLC QDA (MS)

Illustrative results for the hydroxylation of diclofenac by CYP450aluC09 and selected T291x mutants are summarised below in Table 3 with representative chromatograms resented in Figure 3a-c; retention times: diclofenac 1.85-1.88 minutes, 5-hydroxydiclofenac 1.60 - 1.63 minutes, 4’- hydroxydiclofenac 1.65-1.68 minutes, 4’,5-dihydroxydiclofenac 1.40 - 1.42 minutes. The chromatographic retention times and mass spectra coincided with those of authentic samples. The change in the product ratio of 4’- hydroxydiclofenac (2): 5-hydroxydiclofenac (1 ) indicates an altered regioselectivity of aromatic hydroxylation for the catalysis for some of the mutants compared to CYP450aluC09, which is most notable for mutants M24, M33 and M35 showing over four-fold regioselectivity of hydroxylation.

Altered regioselectivity of aromatic hydroxylation is further demonstrated when using other substrates. Albeit of lower overall conversion yield, Table 4 show the changes in regioselectivity of hydroxylation of flufenamic acid, most notably illustrated in mutant M39 showing no detectable production of hydroxyl- flufenamic acid 2, absent at 1.65 minutes compared to the wild-type enzyme CYP450aluC09 and other mutants (Figure 4). Flufenamic acid elutes at 1.93 minutes, hydroxy-flufenamic acid 1 elutes at 1.68 minutes and hydroxy- flufenamic acid 2 elutes at 1.65 minutes.

Table 5 show the changes in regioselectivity of hydroxylation of Aceclofenac, most notably illustrated in mutant M33 showing a reversal of the regioselectivity of hydroxylation of aceclofenac compared to the wild-type enzyme. Chromatograms are provided in Figures 5a-c; aceclofenac elutes at 1.80 minutes, hydroxy-aceclofenac acid 1 elutes at 1.64 minutes, hydroxy- aceclofenac acid 2 elutes at 1.60 minutes and dihydroxy-aceclofenac elutes at 1.42 minutes.

Despite the overriding reaction being the de-A/-ethylation of the napropamide substrate, Table 6 show changes in regioselectivity of hydroxylation pattern of napropamide, most notably illustrated in mutants M34 and M38, showing changes in the ratio both downwards (1.1) and upwards (3.9), respectively, compared to the wild-type enzyme hydroxylation ration (2.1 ) whilst maintinging similar yield. Example chromatograms are provided in Figure 6. Napropamide elutes at 1.76 minutes, unidentified metabolite (-2Da) elutes at 1.90 minutes, desethyl-napropamide elutes at 1.62 minutes, hydroxy- napropamide 1 elutes at 1.53 minutes and hydroxy-napropamide 2 elutes at 1.52 minutes and the desethyl-hydroxy-napropamide product elutes at 1.41 minutes.

Further, the various mutants maintained ability to de-O-methylate indomethacin, as shown in Table 7 to varying degrees. Example UV and MS chromatograms are provided in Figure 7.

Structural formulae of some of the substrates and products described above are provided in Figure 8.

Furthermore, some of the mutants showed greater extent of reaction compared to the wild-type enzyme CYP450aluC09. Table 8 shows the extent of reaction for various additional substrates. The results demonstrate the influence the changing of the amino acid at residue T291 has on extent of substrate metabolism; the extent of reaction for a given mutation is substrate-dependant, no one mutation provides an improved conversion across the set of test substrates. However, of particular note when considering P450 concentration are the results for mutants M12, M20 and M34, as well as the deleterious impact on the range of substrate utility in mutants M39 and M40.

Table 3: Hydroxylation of diclofenac catalysed by cytochrome P450 aluC09 mutants aluC09M11 , aluC09M12, aluC09M17, aluC09M18, aluC09M19, aluC09M20, aluC09M24, aluC09M30, aluC09M31 , aluC09M33, aluC09M34, aluC09M35, aluC09M36, aluC09M37, aluC09M38, aluC09M39, aluC09M40 compared to P450 aluC09 wild-type, as determined by UPLC-PDA-MS. Parent substrate and resulting products have altered UV absorption spectra due to the introduction of phenolic hydroxylation, therefore having different molar absorptivity at the selected wavelength; therefore percentages are for illustration purposes only. Table 3

Table 4: Hydroxylation of flufenamic acid catalysed by cytochrome P450 aluC09 mutants aluC09M11 , aluC09M12, aluC09M17, aluC09M18, aluC09M19, aluC09M20, aluC09M24, aluC09M30, aluC09M31 , aluC09M33, aluC09M34, aluC09M35, aluC09M36, aluC09M37, aluC09M38, aluC09M39, aluC09M40 compared to P450 aluC09 wild-type, as determined by UPLC-PDA-MS. Parent substrate and resulting products have altered UV absorption spectra due to the introduction of phenolic hydroxylation, therefore having different molar absorptivity at the selected wavelength; therefore percentages are for illustration purposes only.

Table 4

Table 5: Hydroxylation of aceclofenac catalysed by cytochrome P450 aluC09 mutants aluC09M11 , aluC09M12, aluC09M17, aluC09M18, aluC09M19, aluC09M20, aluC09M24, aluC09M30, aluC09M31 , aluC09M33, aluC09M34, aluC09M35, aluC09M36, aluC09M37, aluC09M38, aluC09M39, aluC09M40 compared to P450 aluC09 wild-type, as determined by UPLC-PDA-MS. Parent substrate and resulting products have altered UV absorption spectra due to the introduction of phenolic hydroxylation, therefore having different molar absorptivity at the selected wavelength; therefore percentages are for illustration purposes only.

Table 5

Table 6: Hydroxylation of napropamide catalysed by cytochrome P450 aluC09 mutants aluC09M11 , aluC09M12, aluC09M17, aluC09M18, aluC09M19, aluC09M20, aluC09M31 , aluC09M33, aluC09M34, aluC09M35, aluC09M36, aluC09M37, aluC09M38, aluC09M39, aluC09M40 compared to P450 aluC09 wild-type, as determined by UPLC-PDA-MS. Parent substrate and resulting products have altered UV absorption spectra due to the introduction of phenolic hydroxylation, therefore having different molar absorptivity at the selected wavelength; therefore percentages are for illustration purposes only.

Table 6

Table 7: O-demethylation of indomethacin catalysed by cytochrome P450 aluC09 mutants aluC09M11 , aluC09M12, aluC09M17, aluC09M18, aluC09M19, aluC09M20, aluC09M24, aluC09M30, aluC09M31 , aluC09M33, aluC09M34, aluC09M35, aluC09M36, aluC09M37, aluC09M38, aluC09M39, aluC09M40 compared to P450 aluC09 wild-type, as determined by UPLC-PDA-MS. Parent substrate and resulting product have altered UV absorption spectra due to the resulting phenolic hydroxyl having different molar absorptivity at the selected wavelength; therefore percentages are for illustration purposes only.

Table 7

Table 8: Metabolism of various substrates catalysed by cytochrome P450 aluC09 mutants aluC09M11 , aluC09M12, aluC09M17, aluC09M18, aluC09M19, aluC09M20, aluC09M21 , aluC09M24, aluC09M30, aluC09M31 , aluC09M33, aluC09M34, aluC09M35, aluC09M36, aluC09M37, aluC09M38, aluC09M39, aluC09M40 compared to P450 aluC09 wild-type, as determined by UPLC-PDA- MS. Values are percent of each substrate being converted.

Table 8

nt: substrate not tested or no validated test data available

5