In silicoamino acid analysis of ChoA variants

For the identification of a novel bacterial cholesterol oxidase, a Protein Blast search was performed using the cholesterol oxidase amino acid sequence from Streptomyces sp. (UniProt accession number P12676; PDB code 2GEW) as template. Protein sequences of ChoA were retrieved from public databases, aligned using the ClustalW algorithm of the MegAlign software (LASERGENE, Madison, USA), and analyzed in order to identify conserved residues possibly important for the catalytic activity. Out of numerous homologues, the gene choA encoding a hypothetical protein (CgChoA) annotated as cholesterol oxidase was found in the fully sequenced genome of Chryseobacterium gleum ATCC 35910 (DSM 16776; UniProt accession number D7VYA1). The gene was selected for cloning and recombinant expression in E. coli.

The amino acid sequence of CgChoA carries the typical sequence of the Rossmann fold (xh)₂GxGxxGx(xxh)₂(x)₈hxhE, where x is any amino acid and h an hydrophobic one, between V44 and E70 in the N-terminal region. This indicated that CgChoA is an FAD-binding protein [2]. Alignment to selected well-studied cholesterol oxidases and phylogenetic analysis indicated a higher similarity of CgChoA to the non-covalent FAD-dependent enzymes belonging to the Class I family (Figure 2). The lack of a signal peptide indicated the intracellular localization of the enzyme in the native host. Using sequence alignment, CgChoA was analyzed for the presence of residues reported to be important for the catalytic activity [4]. More in detail, residues N485 and Y446 (N522 and Y483 in the original protein sequence GenBank: AAA26719.1, respectively) reported to contribute to the stabilization of the cofactor in the reduced form in the cholesterol oxidase from Streptomyces sp. SA-COO were found conserved in CgChoA, e.g. N503 and Y464. Similarly, amino acid E398, corresponding to E361 (E402) in the cholesterol oxidase from Streptomyces sp. SA-COO, that is supposedly involved in the catalytic process by facilitating deprotonation of the substrate was conserved in CgChoA.

The cDNA sequence encoding CgChoA was cloned into the expression vector pQE-30 such that the final construct pCgChoA coded for an N-terminal His-tag MRGSHHHHHHGSAC fused to CgChoA. The wild-type CgChoA amino acid sequence (without the His-tag) of C. gleum DSM 16776 (UniProt D7VYA1, 528 aa) showed 46.1% identity to that from Streptomyces sp. (PDB code 2GEW, 540 aa), 42.8% identity to that from B. sterolicum (UniProt P22637), 16.1% to that from Mycobacterium tuberculosis (PDB code 2XKR, 398 aa) and 14.1% to that from Chromobacterium sp. (PDB code 3JS8, 587 aa). The CgChoA cholesterol oxidase with the N-terminal His-tag consists of 541 amino acids and has a hypothetical molecular mass of 60.4 kDa.

Expression of cholesterol oxidase from C. gleum choA in E. coli

The gene choA from C. gleum DSM 16776 contains 8% rare codons with respect to the codon usage of E. coli. Therefore, the expression host E. coli JM109 was additionally transformed with the pRARE2 plasmid, which encodes extra copies of genes coding for tRNAs recognizing the codons AGG, AGA, AUA, CUA, CCC, GGA and CGG. E. coli JM109 cells producing CgChoA in the absence of pRARE2 showed only low activity. In the presence of pRARE2, the choA gene was expressed at 30°C, but the protein was found in inclusion bodies. Activity could only be detected in the insoluble fractions. Only when the cultivation temperature was decreased to 16°C immediately after induction, soluble and active protein was present.

Protein purification and characterization

Protein purification was carried out using a Ni-affinity chromatography and subsequently a size exclusion chromatography step. The apparent molecular mass of the expressed CgChoA was around 60 kDa, when visualized on a SDS-polyacrylamide gel (Figure 3). Yields of around 0.2 mg/L culture of purified and enriched CgChoA were usually obtained. Protein bands obtained in SDS-PAGE were analyzed by tryptic digestion, subsequent MS analyses, and in silico processing using Mascot search program (Functional Genomics Center Zürich). The band above 55 kDa was confirmed to be CgChoA by tryptic digest and MALDI-MS/MS. The lower band of approximately 30 kDa of lane 3 (Figure 3) was found to be a houskeeping transferase and isomerase as proven by tryptic digestion and MALDI MS/ESI-MS. The molecular mass of the native protein CgChoA in solution was estimated to be about 85 kDa by size exclusion chromatography on a Superdex 200 pg column. The estimated mass was somewhat higher than 60 kDa, but lower than for a theoretical dimer with 120 kDa, which indicates that the functional enzyme is rather a monomer than a dimer in solution.

Purified CgChoA had a yellow colour and its spectrum showed the characteristic absorbance peaks of flavin-binding proteins (Figure 3). Heat-treatment was used to assess the possible covalent binding of the flavin cofactor to CgChoA apoprotein [16]. The purified enzyme sample after size exclusion chromatography was boiled in the dark for 5 min and centrifuged. A spectrum of the supernatant was recorded between 260–700 nm and showed a typical pattern of an FAD spectrum with two absorption maxima at 370 nm and 470 nm [17]. Only FAD that is non-covalently associated with the enzyme is detectable by this method, as covalently bound FAD co-precipitates with the protein [16].After Ni-affinity chromatography, the partially purified protein was subjected to a pH-screen for best activity in different buffers. First, various buffers were tested as shown in Figure 4 (A). As the enzyme performed using 0.11 M MOPS buffer, this buffer was tested between pH 6–10 and at molarities between 0.55 M and 0.011 M. It was found that cholesterol oxidase activity in the coupled assay was highest using 0.011 M MOPS at pH 6.75, as shown in Figure 4 (B). All subsequent measurements were therefore performed in this buffer. A temperature dependency study was also performed in a similar way. CgChoA maximum activity was measured at around 35°C (Figure 4C). The pH, molarity and temperature screens were performed with cholesterol oxidase from different purification batches that had been stored for different periods prior to use. Calculated volumetric activities as presented in Figure 4 can therefore not be compared directly. However, the overall trend is valid.

The cholesterol-oxidizing activity of purified CgChoA was assayed at 35°C using 0.011 M MOPS, pH 6.75 buffer in a horseradish peroxidase (HRP) coupled assay. 23 cholesterol solutions from 0.17 μM to 5.5 mM were prepared and CgChoA initial activity was determined. We tested ABTS, pyrogallol red and o-dianisidine as hydrogen peroxidase substrates and found only minor changes. However, the amount of co-solvent had a significant influence. As control also E. coli JM109 cells transformed with the pQE-30 vector as empty vector control were tested and additionally the E. coli JM109 transformed with pCgChoA after incubation and induction with IPTG as described. After lysis of the cells no conversion of cholesterol could be detected in the empty vector control. No Michaelis-Menten behaviour was found for CgChoA preparations using cholesterol prepared and diluted in: only water, water with Triton X-100, and water with Triton X-100 and taurocholate, and in these cases and sigmoidal-like curve was obtained when plotting the data obtained (Figure 5). When the substrate was prepared and diluted in water and taurocholate as sole surfactant, a Michaelis-Menten like curve could be fitted and an apparent kinetic constant K_m of 0.5 mM was obtained. For the cholesterol dispersions diluted in water only, a bell-shape profile of the data between 0–0.125 mM cholesterol (see also inset Figure 5) may indicate an activation/deactivation at a low concentration of substrate. A similar activation pattern was found when using a dilution of cholesterol stock solution containing Triton X-100 and taurocholate in water or in water/Triton X-100 and nonionic surfactants and bile acid salts have been described to affect the kinetic behavior at particular enzyme to surfactant ratios [18]. The highest recorded specific activity for CgChoA was 15.5 U/mg.

Enzymatic conversion of cholesterol to cholest-4-en-3-one

Biocatalytic reactions were carried out using purified cholesterol oxidase and cholesterol at a concentration of 1 mM in the presence of 5% v/v Triton X-100. After 42 hours reaction time the product was extracted from the entire reaction batch with chloroform and analyzed. Figure 6 shows the traces monitored by HPLC-DAD at 200 and 250 nm for the enzymatic reaction. The product cholest-4-en-3-one (3), but not cholesterol (1) shows an absorbance at 250 nm. The peak of the chromatogram at 14.4 min at 200 nm corresponds to cholesterol with a mass signal of m/z 369.2 [M-H₂O + H]⁺. The peak of the chromatogram at 13.1 min at 200 and 250 nm corresponds to cholest-4-en-3-one (3) with a mass signal of m/z 385.1 [M + H]⁺ and was only found in the reaction batch which contained cholesterol oxidase. Signals at 4.5 min derived from Triton X-100. There the mass pattern typical for PEG derivatives (series of mass signals differing in Δm/z 44) was observed. The HPLC-MS analysis was performed for qualitative detection of the cholesterol conversion by CgChoA. Additional background signals could not be assigned to relevant compounds by MS. Commercially available cholesterol and cholest-4-en-3-one were used as reference substances.

Bacterial strains

Chryseobacterium gleum DSM 16776 was obtained from the German collection of microorganisms (DSMZ). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(r_K^-, m_K⁺), relA1, supE44, λ^-, Δ(lac-proAB), (F', traD36, proAB, lacI^qZΔM15)] and the pQE-30 expression vector were purchased from Promega (Madison, USA) and Qiagen (Valencia, USA), respectively. The origin of replication in pQE-30 is ColE1 (pBR322) and transcription of the inserted gene is controlled by the bacteriophage T5 promoter (recognized by the E. coli housekeeping RNA polymerase) and two lac operator sequences (conferring inducibility by IPTG). For efficient repression the host strain JM109 which over-expresses the LacI repressor was used. JM109 was transformed with the plasmid pRARE2, which contains the tRNA genes argU, argW, ileX, glyT, leuW, proL, metT, thrT, tyrU, thrU and argX. The usage of the rare codons AGG, AGA, AUA, CUA, CCC, GGA and CGG is thereby supplemented. The plasmid was isolated from Rosetta2 (DE3) (Merck Chemicals, UK) (F^-ompT hsdS_B(r_B^- m_B^-) gal dcm (DE3) pRARE2) cells. The resulting chloramphenicol-resistant strain JM109-pRARE2 was the expression host.

Cloning of choA from C. gleum

The putative cholesterol oxidase gene choA of C. gleum was identified by Protein blast (NCBI website) using the cholesterol oxidase sequence of Streptomyces sp. (UniProt accession no. P12676) as search template. The cholesterol oxidase gene of C. gleum (accession no. ACKQ02000004) was PCR amplified from genomic DNA with forward primer 5’ GCG GCA TGC GAC AGA AAA AAA TTC ATC AGG ACA AGT GC 3’ (introducing a SphI site around the native start codon) and reverse primer 5’ CCG AAG CTT TTA ACC CAG GTT AAA TTC ATT TTG CCG G 3’ (introducing a HindIII site after the native stop codon). PCR was performed with high fidelity Phusion polymerase (New England Biolabs, Ipswich, USA) and a diluted solution of genomic DNA of C. gleum DSM 16776 as template source. Genomic DNA was isolated using the GenElute Bacterial genomic DNA kit (Sigma-Aldrich, CH). Plasmid DNA and PCR products were purified using the Gene Jet Plasmid Miniprep Kit (Fermentas) and the GenElute PCR clean-up kit (Sigma-Aldrich). DNA from agarose gels was recovered using the GenElute Gel extraction kit (Sigma-Aldrich, CH). The 1596 bp PCR product was cloned into the pQE-30 expression vector in frame with a sequence coding for an N-terminal hexa-histidine tag to allow purification by immobilized metal affinity chromatography. The in frame cloning of the choA gene from C. gleum DSM 16776 in the final expression plasmid pCgChoA was confirmed by DNA sequencing (GATC, Germany).

Cell cultivation and protein purification

C. gleum DSM 16776 was grown overnight at 30°C at 180 rpm in trypticase soy yeast extract medium (trypticase soy broth 30 g/L, yeast extract 3 g/L). E. coli JM109-pRARE2 was transformed with pCgChoA. Expression of the recombinant protein was performed in medium containing 1× M9 salts, 20 g/L N-Z-amine, 20 g/L glycerol, 1 mM MgSO_4, 1 mM MgCl₂, 100 μM CaCl₂, 100 μM thiamine, 0.025% glucose and trace metal mixture [29]. A 100 mL overnight culture was grown from a single colony (LB agar) and used to inoculate 700 mL of medium (dilution 1:50). The culture was grown at 37°C with shaking at 180 rpm. At an OD₆₀₀ = 0.8, protein production was induced at 0.1 mM isopropyl thio-β-D-galactoside (IPTG). At the same time, the temperature and shaking were reduced to 16°C and 120 rpm for 16–18 hours. For plasmid selection 100 μg/mL ampicillin and 20 μg/mL chloramphenicol were added to plates and liquid media. For protein purification cells were harvested by centrifugation at 4°C for 30 min at 4,495 × g, washed in 0.1 M sodium phosphate buffer pH 7, centrifuged again and subsequently stored at -20°C. Frozen cells were thawed on ice and resuspended in 0.1 M sodium phosphate buffer pH 7 with 20 mM imidazole and 0.5 M sodium chloride (buffer A) containing 1 mg/mL lysozyme and protease inhibitor mix (Roche Complete Protease Inhibitor Mix, EDTA-free) and re-frozen at -80°C. Cells were thawed, Benzonase Nuclease (Roche) was added and the suspension incubated for 1 h at 37°C at 120 rpm. The suspension was subjected to twelve 10 s rounds of sonication with a Branson sonicator equipped with a microtip at a setting of 80%. Cellular debris was removed by centrifugation at 4°C for 40 min, 47,000 × g. Purification was performed on an Äkta purifier FPLC system (GE-Healthcare). The sample was loaded onto a 1 mL HisTrap FF chromatography column (GE-Healthcare), previously equilibrated with buffer A. Proteins were eluted with a imidazole gradient from 0 to 1 M. Fractions displaying cholesterol activity were pooled and concentrated by ultrafiltration using a 30 kDa cut-off. The sample was loaded onto a Superdex 200 column (GE-Healthcare), previously equilibrated with 20 mM MOPS buffer pH 6.75 containing 0.1 M NaCl. Fractions with cholesterol oxidase activity were pooled and concentrated by ultrafiltration. The purity of the sample was analyzed by SDS-PAGE using a 10% polyacrylamide gel. The gel filtration kit (GE-Healthcare) was used to calibrate a Superdex 200 column with high and low molecular weight standards, previously equilibrated with 20 mM MOPS buffer (pH 6.75) containing 0.1 M NaCl.

Activity assay and protein determination

A 27.2 mM stock solution/dispersion of cholesterol was prepared and diluted in water in the presence or absence of 5% (v/v) Triton X-100, 2.9% (w/v) of taurocholic acid sodium salt (Sigma Aldrich), and a combinations thereof. Cholesterol oxidase activity was assayed by quantifying H₂O₂ formation from the coupling reaction with HRP. The activity assay mixture contained 40 μL of cholesterol at the selected concentration, 10 μL of HRP (concentration 1 mg/mL, in ddH₂O), 10 μL of ABTS (concentration 10 mM, in ddH₂O), 110 μL of 0.011 M MOPS buffer pre-heated to 37°C, and 30 μL of the purified enzyme preparation in a total volume of 200 μL. The spectrophotometric cholesterol activity assay was carried out in a 96-well plate using a BioTek Synergy Mx spectrophotometer. ABTS (0.6 mM), pyrogallol red (0.15 mM) and o-dianisidine (0.5 mM) were used as substrates for the HRP coupled assay using 0.011 M MOPS buffer pH 6.75 at 37°C. The reaction was started by adding cholesterol oxidase and followed for oxidation of ABTS at 420 nm (ε = 36 000 M^-1 cm^-1), of pyrogallol red at 550 nm (ε = 30 900 M^-1 cm^-1) and of o-dianisidine at 440 nm (ε = 13 000 M^-1 cm^-1). Kinetic parameters of cholesterol oxidase samples were determined between 0.17 μM – 5.5 mM cholesterol at 35°C, and results were analyzed with the Enzyme Kinetics Module of the software SigmaPlot (Systat Software Inc., CA, USA).

Cholesterol activity as a function of the pH was recorded via the HRP coupled assay with 0.5 mM ABTS and 0.55 mM cholesterol using Teorell-Stenhagen buffer (pH 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, and 8.5), 0.1 M sodium phosphate buffer (pH 6.0, 6.4, 7.0, 7.5, and 7.8), 0.11 M MOPS pH 6.75, 0.1 M potassium phosphate buffer (pH 6.0, 6.5, 7.0, 7.5, and 7.8), and McIlvaine buffer (pH 4.0, 5.0, 6.0, 7.0, 7.5, and 8.0). Further 0.55 M, 0.275 M, 0.11 M 0.055 M, 0.0275 M and 0.011 M MOPS buffers (pH 6.75, 7, 7.25, 7.5, 7.75, and 8.0) were tested. The temperature optimum was recorded between 24 and 48°C following ABTS oxidation with 0.55 mM cholesterol, in an assay volume of 3 mL using a magnetically stirred, temperature-controlled cuvette device using a Varian Cary 50 Bio spectrophotometer. Total protein concentration was determined by the method of Bradford, with bovine serum albumin as standard.

Biocatalytic reactions

The substrate cholesterol was added from a stock solution, which was made up as described above (containing Triton X-100 and taurocholate), to a final concentration of 1 mM in 0.011 M MOPS buffer pH 6.75. The reaction was adjusted to 600 μL and 0.04 mg of purified cholesterol oxidase from C. gleum was added (0.67 U/mL). For the blank reaction water was used instead of enzyme solution. All reactions were prepared in triplicate. The reaction mixture was left shaking at 250 rpm at 30°C for 42 hours in 3 mL screw cap glass vials.

Analysis of cholesterol and cholest-4-en-3-one by HPLC-MS

The total reaction was extracted on 1 mL chloroform. After evaporation of the solvent at room temperature, the product was dissolved in the solvent, which was the same as the mobile phase used for HPLC. 10 μL of the analyte sample were injected into a Phenomenex Gemini® 5 μ C18 110 Å column (250 × 4.6 mm, 5 micron), and chromatography under isocratic conditions was performed using methanol:water 100:2 (v/v) at a flow rate of 0.8 mL/min at room temperature. Cholesterol and cholest-4-en-3-one were purchased from Sigma-Aldrich and used as reference. Product formation was monitored at 200 and 250 nm, whereas cholesterol was detected at 200 nm. The Agilent HPLC 1100 system equipped with a DAD was coupled to an esquireHCT ion trap mass spectrometer (Bruker. Germany), and an atmospheric pressure chemical ionization (APCI) source was operated in the positive ion mode. Conditions were as follows: scan range, m/z 50–600; dry gas flow of 11 L/min, nebulizer pressure 35 psi, drying gas temperature 320°C and the APCI heater temperature was 350°C. The extracted ion current (EIC) signals were deduced based on the exact masses for protonated cholesterol after water elimination (m/z 369.34) as well as for the protonated oxidation product cholest-4-en-3-one (m/z 385.34).