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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
  • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
  • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
  • C12Y110/03011—Ubiquinol oxidase (1.10.3.11)

Definitions

• the present invention relates to alternative oxidases, and in particular to plant, fungal and bacterial recombinant alternative oxidases.
• the invention is concerned with genetic constructs comprising a coding sequence encoding alternative oxidase, and extends to a method of producing highly active and pure recombinant alternative oxidase.
• the alternative oxidase is a non-proton motive ubiquinol oxido- reductase, which catalyzes the 4-electron reduction of dioxygen to water.
• Genes encoding AOX have been found in higher plants, algae, fungi, yeast, slime molds, free-living amoebae, eubacteria, nematodes and protists.
• bioinformatic searches have broadened the taxonomic distribution of AOX to some members of the animal kingdom.
• AOX AOX-oxide-semiconductor
• AOXs also play a role in the metabolism of a multitude of other organisms. Its ubiquitous nature may suggest that the metabolic flexibility that the alternative pathway confers upon an organism permits it to respond to a wide range of developmental and environmental conditions.
• Electron Paramagnetic Resonance EPR
• FTIR Fourier Transform Infrared
• a method of preparing recombinant Alternative Oxidase comprising culturing a host cell comprising a genetic construct comprising a nucleic acid molecule which encodes an alternative oxidase (AOX) under conditions suitable for the production of rAOX; and contacting the rAOX with a keto acid.
• the method of the invention enables the preparation of purified rAOX, which is highly active and exhibits exceptional stability upon storage. Furthermore, kinetic characterization of the rAOX has revealed that it exhibits typical Michaelis-Menten kinetics and is potently inhibited both by
• keto acid e.g. pyruvate
• keto acid such as pyruvate
• a keto acid can be an organic acid which contains a carboxylic acid group and a ketone group. In some cases, the keto group maybe hydrated.
• the keto acid maybe an alpha-keto acid (i.e. 2-oxoacid), a beta-keto acid (i.e. 3-oxoacid) or a gamma-keto acid (i.e. 4- oxoacid).
• the keto acid is an alpha-keto acid.
• the keto acid maybe pyruvic acid (i.e. pyruvate), oxaloacetic acid or glycolic acid.
• the keto acid is pyruvate.
• the concentration of keto acid contacted with the rAOX may be at least imM, 2mM, 5mM or 8mM.
• the concentration of keto acid is i2mM or lomM or less.
• the concentration of keto acid maybe between imM and lomM, or between 2mM and lomM, or between 5mM and lomM.
• the keto acid may form part of a buffered medium.
• the medium may comprise Tris-HCl.
• the pH of the buffered medium maybe between 7.0 and 8.0.
• the method may comprise contacting the rAOX with an emulsifying agent.
• the rAOX maybe contacted with the emulsifying agent after it has been contacted with the keto acid.
• a suitable emulsifying agent maybe EDT-20.
• concentration of the emulsifying agent maybe at least 0.01% (v/v), 0.02% (v/v) or at least 0.025% (v/v).
• concentration of emulsifying agent is 0.025% (v/v) or less.
• concentration of emulsifying agent maybe between 0.01% and 0.025% (v/v), or between 0.02% and 0.025% (v/v).
• an emulsifying agent such as EDT-20, stabilises the rAOX in its active-form, which the inventors believe may be as a result of mimicking the mitochondrial membrane.
• the host cell maybe a yeast cell, a fungal cell or a bacterial cell, for example E. coli.
• the host cell may be a heme-deficient strain, such as a heme-deficient E. coli strain.
• the host cell may be E. coli AAhemA mutant (FN102) strain, which lacks quinol oxidase activity of the cytochrome bo and bd complexes.
• this strain of E. coli lacks some enzymes that would normally allow the E.
• the expression of the rAOX rescues the host organism by allowing it to synthesise a terminal oxidase, thereby enabling the functional expression of AOX separate from other terminal oxidases.
• the host cell maybe E .coli C4i(DE3), as described in Miroux and Walker, 1996, Journal of Molecular Biology, 260 289-298. This strain is useful as a host when other terminal oxidases in addition to the AOX need to be expressed (for example, in order to test the specificity of agrochemicals targeted at the AOX, or the cytochrome bci complex).
• the host cell may be an animal cell, for example a mouse or rat cell. It is preferred that the host cell is not a human cell.
• the method comprises culturing the host cell, and once the culture has reached optimum cell density, the method may then comprises inducing the cells with a suitable inducing agent which is capable of stimulating expression of the rAOX.
• a suitable inducing agent which is capable of stimulating expression of the rAOX.
• the method may comprise inducing the cells with a suitable inducing agent which is capable of stimulating expression of the rAOX.
• the inducing agent may be Isopropyl-P-D-thio-galactoside (IPTG).
• IPTG Isopropyl-P-D-thio-galactoside
• the optimum concentration of the inducing agent maybe at least ⁇ , 2 ⁇ , 3 ⁇ , or 4 ⁇ .
• the concentration of the inducing agent maybe 5 ⁇ or ⁇ or less.
• the method may comprise incubating the host cell for at least a further 2, 4, 6, 8, 10, 12 or 14 hours to allow expression of the rAOX.
• the host cell may be harvested, for example by centrifugation.
• the method may then comprise re- suspending harvested cell pellets in the presence of the keto acid, such as pyruvate.
• the concentration of keto acid in the re-suspension medium maybe as described above and as used for the growth/ expression steps.
• the method may comprise contacting the host cell with a protease inhibitor.
• a protease inhibitor cocktail maybe that which is known as 'Complete' protease inhibitor cocktail tablets, available from Roche Diagnostic GmbH, Germany, which maybe supplemented with up to lOOmM
• the method may then comprise lysing the host cell to release the rAOX. After lysis, cell debris may be removed, for example by centrifugation. Pellets containing the cell membranes may then be re-suspended in the presence of the keto acid, for example pyruvate, which may be present at a concentration as used for the previous steps.
• the method may comprise solubilising the host cell's cell membrane.
• Solubilisation may comprise contacting the host cell with a suitable detergent or solubilization buffer, which may comprise the keto acid, for example pyruvate.
• the concentration of keto acid in the solubilisation buffer may be as described above.
• the solubilisation buffer may comprise n-Dodecyl-P-D-maltopyranoside (DDM).
• DDM n-Dodecyl-P-D-maltopyranoside
• the solubilisation buffer comprises at least 0.5% (v/v) DDM or at least at 0.8% (v/v) DDM.
• DDM can act as an effective detergent, which the inventors believe to be important for solubilisation of the rAOX.
• one or more subsequent buffer comprising the rAOX also comprises at least 0.5% (v/v) DDM in order to keep the rAOX soluble.
• one or more subsequent buffer comprising the rAOX may further comprise at least 0.5% (v/v) octyl-glucoside (OG).
• one or more subsequent buffer comprising the rAOX may further comprise at least 1 % (v/v) Ci 2 Es
• the method may comprise purifying the rAOX.
• Purification may comprise the use of chromatography, for example affinity chromatography.
• the rAOX may comprise a histidine tag.
• solubilized rSgAOX maybe purified by cobalt or nickel affinity chromatography.
• the rAOX which has been solubilized by DDM may be bound to an affinity resin in the presence of DDM, magnesium sulphate, glycerol, octyl-glucoside and pyruvate.
• the rAOX bound to the resin may be eluted with buffer containing imidazole.
• the rAOX is eluted with buffer containing DDM, magnesium sulphate, glycerol, octyl-glucoside, pyruvate and imidazole.
• the concentration of pyruvate in this step is as described above and as used for the previous steps, and it should be maintained to substantially increase rAOX stability, regardless of detergent concentration. Similarly, preferably about lomM MgSO 4 should be maintained during purification. A minimum of 5% (v/v) glycerol is preferably maintained during purification.
• Purified rAOX maybe obtained by elution with imidazole resulting in a very efficient purification of active enzyme.
• one or more subsequent buffer comprising the purified rAOX may comprise a cross-linker, which is able to stabilize the rAOX.
• a suitable cross-linker may comprise diamide.
• the keto acid e.g. pyruvate
• the keto acid should preferably be added to each subsequent buffer used following the growth phase. If pyruvate is likely to interfere with any downstream experiments or analysis, then it may be desirable to remove it at any point (for example using polytheylene glycol precipitation), and then re-suspending the rAOX in a buffer without pyruvate, or containing only low concentrations thereof. It is preferred that the keto acid (i.e. pyruvate) is removed at the last possible step in the method of the invention, because its removal prior to this may result in low activity and a decrease in rAOX stability.
• the keto acid i.e. pyruvate
• 25omM imidazole coupled to a fraction collector may be used in order to avoid over-dilution of the rAOX in the eluate fractions. Collection tubes containing the highest AOX activities may then be pooled, if required.
• purified rAOX protein is required for crystallography, it is preferred to reduce the glycerol concentration to 5% (v/v) or less in each subsequent buffer used after protein solubilisation.
• a maximum concentration of 0.5% (v/v) DDM is preferred in the or each buffer after solubilisation, and no other detergent should be added.
• Keto acid (e.g. pyruvate) concentration should be maintained, however.
• Protein samples for Western blotting/SDS PAGE analysis maybe concentrated using acetone. Pure protein samples can be precipitated out using drop-wise addition of a 50% polyethylene glycol (PEG) 6000 solution followed by centrifugation. Once the supernatant has been carefully aspirated, the pellet may then be frozen at -8o°C until required, or re-suspended in a smaller volume of buffer if concentration is required.
• PEG polyethylene glycol
• the AOX maybe a eukaryotic or prokaryotic AOX enzyme.
• the AOX maybe a plant AOX, a fungal AOX or a bacterial AOX.
• the nucleic acid sequence encoding AOX enzymes maybe found from the publicly available databases. For example, as described in the Examples, in one embodiment, the AOX maybe Sauromatum guttatum AOX, which is also referred to as T. venosum.
• guttatum AOX (Accession Number: M60330) is provided herein as SEQ ID No: 1, as follows: atgatga gctcgcgtct ggtcggcacc gctctctgca ggcagctcag
• the nucleic acid molecule which encodes the rAOX, may comprise a nucleotide sequence substantially as set out in SEQ ID No:i, or a functional variant or a fragment thereof.
• the polypeptide sequence of 5. guttatum AOX is provided herein as SEQ ID No: 2, as follows.
• the rAOX may comprise an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof.
• Genetic constructs used in the method of the first aspect may be in the form of an expression cassette, which maybe suitable for expression of the rAOX in the host cell.
• the genetic construct may be introduced into the host cell without it being incorporated in a vector.
• the genetic construct which may be a nucleic acid molecule, may be incorporated within a liposome or a virus particle.
• a purified nucleic acid molecule e.g. histone-free DNA, or naked DNA
• suitable means e.g. direct endocytotic uptake.
• Suitable means for introducing the genetic construct into the host cell will depend on the type of cell.
• the genetic construct maybe introduced directly in to cells of the host subject (e.g. a bacterial cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
• genetic constructs of the invention may be introduced directly into a host cell using a particle gun.
• the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell.
• the recombinant vector maybe a plasmid, cosmid or phage. Such recombinant vectors are useful for transforming host cells with the genetic construct, and for replicating the expression cassette therein, such that the rAOX is expressed.
• Suitable backbone vectors include pET-i5b (see Figure 2) to form an expression vector pETSgAOX (see Figure 3).
• Recombinant vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression and/or a suitable terminator to cease gene expression.
• a suitable promoter may be the T7 promoter, and an example of a suitable terminator maybe the T7 terminator.
• the recombinant vector maybe designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication maybe required in the recombinant vector.
• the recombinant vector may be designed such that it integrates into the genome of a host cell.
• DNA sequences which favour targeted integration e.g. by homologous recombination are envisaged.
• the recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA.
• a selectable marker gene may be in a different vector to be used simultaneously with vector containing the gene of interest.
• the vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of the host cell.
• the construct may comprise a nucleotide sequence substantially as set out in SEQ ID No:i, or a functional variant or a fragment thereof and/or encode a rAOX, which comprises an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof.
• rAOX recombinant Alternative Oxidase obtained by, or obtainable from, the method of the first aspect.
• the rAOX of the third aspect maybe stable for at least 1, 2, 3, 4, 5 or 6 months.
• the activity of the rAOX maybe greater than ⁇ , 2 ⁇ 1, or 3 ⁇ 1 QH 2 oxidised per minute per mg protein.
• the oxidation of ubiquinol-i by the rAOX of the invention displayed typical Michaelis-Menten kinetics (K m of 332 ⁇ and V ma x of 30 ⁇ mol ⁇ 1 min ⁇ 1 mg ⁇ 1 ), a turnover number of 20 ⁇ s _1 and remarkable stability.
• the rAOX was stimulated upon contacting with pyruvate (lomM) and EDT-20 (0.025%) indicating that the recombinant enzyme retains biochemical properties similar to the native protein.
• EDT-20 decreased the Km of the rAOX for QiH 2 from 332 ⁇ to ⁇ and increased V ma x to 37.8 ⁇ - 1 mm 1 mg 1 , thereby suggesting that the correct conformational state of the protein is required to achieve maximal activity.
• nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
• amino acid/polynucleotide/polypeptide sequence substantially the amino acid/polynucleotide/polypeptide sequence
• “functional variant” and “functional fragment” can be a sequence that has at least 40% sequence identity with the amino acid/polynucleotide/polypeptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleic acid sequence identified as SEQ ID No:i (i.e. DNA sequence of plant AOX), or 40% identity with the polypeptide identified as SEQ ID N0.2 (i.e. protein sequence of plant AOX), and so on.
• the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
• the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino
• the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith- Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences.
• the method used to align the sequences for example, ClustalW, BLAST, FASTA, Smith- Waterman (implemented in different programs), or structural alignment from 3D comparison
• the parameters used by the alignment method for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.
• percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
• acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
• a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No: 1, or their complements under stringent conditions.
• stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/0.1% SDS at approximately 20-65°C.
• a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequence shown in SEQ ID No: 2.
• nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
• Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
• Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
• small non- polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
• Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
• the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
• the positively charged (basic) amino acids include lysine, arginine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids maybe replaced with an amino acid having similar biophysical properties, and the skilled technician will known the nucleotide sequences encoding these amino acids.
• Figure 2 is a plasmid map of pETisb
• Figure 3 is a schematic representation of one embodiment of an expression construct according to the invention, pET.SgAOX;
• FIG. 4 shows SDS-PAGE analysis of recombinant S. guttatum AOX (rSgAOX);
• Figure 5 shows a Western blot of the SgAOX;
• Figure 6 shows the oxygen uptake by E. coli membranes expressing rSgAOX and the effect of the inhibitor, ascofuranone, on the rate of respiration. Rates are expressed as ⁇ 0 2 consumed/min/mg protein;
• Figure 7 shows the effects of EDT-20 on the specific activity of the rSgAOX of the invention.
• E. coli AhemA mutant (FN102) strain which lacks quinol oxidase activity of the cytochrome bo and bd complexes (Nihei et al., 2003, FEBS Lett. 538: 35-40), was used for the expression of recombinant alternative oxidase (rAOX). - ⁇ 5 -
• S. guttatum AOX lacking a mitochondrial localization signal sequence (SEQ ID No:i) was expressed in E. coli.
• the cleavage sites were predicted using MitoProt (http://ihg2.helmholtz-muenchen.de/ihg/mitoprot.html; M.G. Claros, P. Vincens. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779-786 (1996)).
• a recognition site for Ndel was introduced at the SgAOX cleavage site.
• E. coli (FN102) cells were transformed with the pET.SgAOX construct shown in Figure 3, and grown overnight on selective Luria agar supplemented with amino-levulinic acid (ALA),
• ALA amino-levulinic acid
• ampicillin. FN102 is a slow grower, and has the wrong complement of enzymes to make respiratory proteins.
• This particular strain of E. coli lacks some enzymes that would normally allow the E. coli to grow.
• the expression of AOX rescues the organism by allowing it to synthesise a terminal oxidase. Thus, use of this strain is important for the functional expression of AOX separate from other terminal oxidases.
• a single colony was used to streak a fresh agar plate with the same supplements, and was incubated for 12 hours at 37°C. A scrape of cells from the streak plate was used to inoculate 50ml starter culture (Luria broth, ALA, ampicillin).
• IPTG Isopropyl-P-D-thio-galactoside
• C4i(DE3) E. coli cells were transformed with the pET.SgAOX construct shown in Figure 3, and grown overnight on selective Luria agar supplemented with ampicillin. A single colony was used to streak a fresh agar plate with the same supplements, and was incubated for 12 hours at 37°C. A scrape of cells from the streak plate was used to inoculate 100ml starter culture (Luria broth, lOO ⁇ g/ml ampicillin). The starter culture was grown at 37°C with shaking overnight.
• starter culture Lia broth, lOO ⁇ g/ml ampicillin
• the whole starter culture was then used to inoculate 5L of Luria broth (supplemented with lOO ⁇ gml ⁇ 1 ampicillin, 0.2% glucose, 0.125% FeS0 4 ) which was the incubated at 30°C with shaking until the OD650 reached 0.4.
• the temperature of the incubator was then reduced to i8°C and the culture was incubated with shaking for one hour. After this hour the culture was induced with 25 ⁇ IPTG and incubated at i8°C for 18 hours with shaking.
• E. coli FN102
• E. coli C4i(DE3) E. coli C4i(DE3)
• cells were harvested by centrifugation at 8ooog (6 minutes) and cell pellets were re-suspended in 50mM Tris-HCl, lomM pyruvate, pH 7.5. After the pellets were pooled and homogenised, a protease inhibitor cocktail (Roche "Complete”) and loomM PMSF were added, before lysis using a French Press (10k psi, two passes).
• Membranes were treated with solubilization buffer (6 mg/ml protein in 50 mM Tris-HCl, lomM pyruvate, 1% (w/v) n-Dodecyl-P-D-maltopyranoside (DDM), 20%(v/v) glycerol, pH 7.5) at 4°C and immediately ultracentrifuged at 200,000 g for 1 hr at 4°C. The ubiquinol oxidase activities of the samples before centrifugation, as well as of supernatant and pellet, were then determined.
• solubilization buffer 6 mg/ml protein in 50 mM Tris-HCl, lomM pyruvate, 1% (w/v) n-Dodecyl-P-D-maltopyranoside (DDM), 20%(v/v) glycerol, pH 7.5
• AUC may also be used to determine whether the rAOX is encased in micelles (which may prevent efficient binding to the affinity resin) at any point during the purification process once the sedimentation coefficient has been determined. Purification ofrAOX
• the resin was washed twice with 2.5ml of wash buffer (50 mM Tris-HCl, 20 mM imidazole, 0.5% DDM, 0.5% (w/v) C12E8 (Sigma), lOOmM MgS0 4 , 20% (v/v) glycerol, lomM pyruvate, pH 7.5) and the resin-bound rAOX was transferred to a column.
• wash buffer 50 mM Tris-HCl, 20 mM imidazole, 0.5% DDM, 0.5% (w/v) C12E8 (Sigma), lOOmM MgS0 4 , 20% (v/v) glycerol, lomM pyruvate, pH 7.5
• rAOX was eluted with increasing imidazole concentrations by mixing elution buffer (50 mM Tris-HCl, 250 mM imidazole, 0.5% DDM, 0.5% C12E8, 100 mM MgSO 4 l, 20%(v/v) glycerol, lomM pyruvate, pH 7.5 and wash buffer (as above). 1 ml fractions were collected.
• elution buffer 50 mM Tris-HCl, 250 mM imidazole, 0.5% DDM, 0.5% C12E8, 100 mM MgSO 4 l, 20%(v/v) glycerol, lomM pyruvate, pH 7.5 and wash buffer (as above).
• rSgAOX i.e. a ubiquinol oxidase
• Ubiquinol oxidase activity was measured by recording the change in absorbance of ubiquinone-i (i.e. absorbance increases as ubiquinol-i is oxidised to ubiquinone-i) at 278 nm (Varian Cary 4000 spectrophotometer).
• EDT-20 also known as PEG-10 Tallow
• Respiratory activity of the rSgAOX was measured with a Clark-type electrode (Rank Brothers, Cambridge, U.K.) using 0.1 to 0.5 mg E. coli membranes suspended in 0.4 mL air-saturated reaction medium (250 ⁇ at 25 °C, R.R. Wise, A.W. Naylor, Calibration and use of a clark-type oxygen-electrode from 5 to 45 °C, Anal. Biochem. 146 (1985) 260-264) containing 50 mM Tris-HCl (pH 7-5).
• Colletochlorin B was synthesised using the technique described by K.-M. Chen and M. M. Joullie (Tetrahedron letters, 23, 4567-4568, 1982 Synthesis of Colletochlorine B), using geranyl bromide as the alkylating agent in the final step, resulting in a white product (20%).
• membrane-bound recombinant protein For large-scale drug-protein interaction screening, using membrane-bound recombinant protein is recommended due to increased protein life-span and stability. Membrane-bound protein is also easy to assay using either an oxygen electrode or a spectrophotometric assay following the oxidation of NADH. This will also reduce the bottle-neck encountered with the purification of the recombinant alternative oxidase. For further analysis of selected drugs purified protein maybe used. If membrane-bound protein is indeed required, then all steps prior to solubilisation should be used.
• Oligonucleotides were obtained from MWG Biotech. Sequencing was performed by Beckman Coulter Genomics. Other procedures were as described by
• Sauromatum guttatum AOX (rSgAOX), and purification protocols were significantly optimized in order to obtain large quantities of active and stable rSgAOX.
• the presence of pyruvate was believed to important as it causes a conformational change in the rSgAOX, which, as discussed below, caused a surprising increase in the overall activity of the enzyme.
• the pyruvate also stabilises the enzyme by some unknown mechanism.
• EDT-20 as it stabilises the enzyme in its active-form, possibly by mimicking the mitochondrial membrane.
• the presence of pyruvate and EDT-20 were believed to be important but for somewhat different and unknown reasons.
• the purified rSgAOX with a molecular mass of 34 kDa, was estimated to be at least 97% pure by SDS-PAGE, as shown in lane 4 of Figure 4.
• other bands could also be identified including two bands with a smaller size than rSgAOX and one band with an approximate molecular mass of 74 kDa. Since all of these bands were recognized in the Western blot using a monoclonal antibody against SgAOX (see Figure 5), the smaller protein bands possibly represent proteolytic breakdown products whilst the 74 kDa band represents a dimer of rSgAOX.
• the specific activity of the purified rSgAOX was found to be more than 30 ⁇ " 1 min t ing- 1 when 150 ⁇ of ubiquinol-i was used as a substrate in the presence of lomM pyruvate.
• Quinol oxidase activity, of purified rSgAOX, as measured by the oxygen electrode (see Figure 6) was insensitive to 1 mM KCN or luM antimycin A, but was completely inhibited by 10 nM ascofuranone, as shown in Figure 6. It should be noted that Figure 6 shows the rate in ⁇ O 2
• oxidised/min/mg protein As summarized in Table 1, a greater than 22-fold increase in purification was achieved using the techniques described above, and 49.1% of the total activity was recovered from the lysate of FNi02/pSgAOX cells. Such procedures resulted in approximately 5 mg of highly purified rSgAOX from a 5 1 culture.
• Table 2 indicates the sensitivity of the purified rSgAOX protein to a range of AOX inhibitors including SHAM, n-propyl gallate, ascofuranone and
• colletochlorine B are much more potent than the other AOX inhibitors with IC 50 values ranging from 5-2onM.
• Trypanosome Alternative Oxidase (TAO) and Arum italicum AOX reported that the addition of a specific detergent (0.025% EDT-20) during the assay increased the activity 3- to 4-fold close to saturation.
• Vmax ⁇ mol/min/mg) 37.8 30.2 kcat 22.5 /sec
• rSgAOX recombinant SgAOX
• FN102 AhemA-deficient Escherichia coli strain
• E. coli membranes were solubilized and purified to homogeneity in a very stable and active form.
• SDS gels and Western blots revealed a doublet band located at approximately 32kDa.
• the oxidation of ubiquinol-i, by purified rSgAOX, displayed typical Michaelis-Menten kinetics (K m of 332 ⁇ and V ma x of 30 ⁇ " ! min- ! mg 1 ), a turnover number of 20 ⁇ s 1 and remarkable stability.
• the purified recombinant protein was stimulated by lomM pyruvate (which is a keto acid) and 0.025% EDT20 similar to that observed with the protein isolated from thermogenic plants indicating that the recombinant protein retains biochemical properties similar to the native protein.
• the pyruvate is believed to cause a conformational change in the rSgAOX, which results in a surprising increase in the overall activity of the enzyme.
• the pyruvate also stabilises the enzyme.
• the EDT-20 stabilises the enzyme in its active-form, possibly by mimicking the mitochondrial membrane.

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