Báo cáo khoa học: Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacteriumsp. strain HE5

Kinetic and Binding Studies with Purified Recombinant Proteins Ferredoxin Reductase, Ferredoxin and Cytochrome P450 Comprising the Morpholine Mono-oxygenase from Mycobacterium sp. Strain HE5

Tác giả: Bernhard Sielaff and Jan R. Andreesen

Lĩnh vực: Institut für Mikrobiologie, Martin-Luther-Universität Halle, Germany

Nội dung tài liệu:

Nghiên cứu này tập trung vào hệ thống enzyme P450mor từ vi khuẩn Mycobacterium sp. strain HE5, được cho là có vai trò trong quá trình hydroxyl hóa các hợp chất N-heterocyclic. Hệ thống này bao gồm ba thành phần chính: ferredoxin reductase (FdRmor), ferredoxin (Fdmor) và cytochrome P450 (P450mor). Nghiên cứu đã thực hiện việc tinh sạch các protein Fdmor và P450mor dưới dạng tái tổ hợp, cũng như FdRmor đã được phân lập trước đó. Các phân tích động học về cặp redox FdRmor/Fdmor đã cho thấy sự ưu tiên rõ rệt đối với việc khử nitroblue tetrazolium (NBT) phụ thuộc NADH và sự cần thiết tuyệt đối của Fdmor trong phản ứng này, so với việc khử cytochrome c phụ thuộc NADH. Hằng số Michaelis (Km) thấp của FdRmor đối với Fdmor, được đo bằng NBT làm chất nhận electron, cho thấy độ đặc hiệu cao. Việc bổ sung các chuỗi His-tag ở đầu N hoặc C của Fdmor không làm thay đổi đáng kể các thông số động học, nhưng lại dẫn đến hoạt tính nền cạnh tranh. Việc sản xuất P450mor dưới dạng protein dung hợp His-tag ở đầu N đã cho phép tinh sạch protein ở dạng hoạt động quang phổ, điều mà trước đây chưa thực hiện được với P450mor dạng tự nhiên. Các chất nền tiềm năng như morpholine, piperidine hoặc pyrrolidine không tạo ra phổ liên kết chất nền với P450mor trong mọi điều kiện thử nghiệm. Tuy nhiên, pyridine, metyrapone và các hợp chất azole khác đã tạo ra phổ liên kết loại II, và các giá trị Ka xác định được cho thấy P450mor có thể ưu tiên các phân tử cồng kềnh và/hoặc ưa mỡ hơn. Các protein tái tổ hợp FdRmor, Fdmor và P450mor đã được sử dụng để tái tạo hoạt động của mono-oxygenase chứa P450 tương đồng, và hệ thống này đã được chứng minh là có khả năng chuyển hóa morpholine.

Mục lục chi tiết:

• Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5
• Keywords
• Correspondence
• The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor).
• In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously.
• Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c.
• The quite low Km (5.3 ± 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity.
• The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins.
• Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor.
• The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions.
• Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules.
• The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.
• P450 cytochromes are well known for their involvement in the synthesis of various antibiotics in different Streptomyces species [1-4].
• But they also account for many of the various degradative abilities on xenobiotic compounds, which have been reported for other Actinomycetales [5-9].
• The involvement of a cytochrome P450 in the degradation of the secondary cyclic amines morpholine, piperidine and pyrrolidine has been shown for different Mycobacterium species [10-14].
• A P450-containing mono-oxygenase was supposed to catalyse the initial hydroxylation of these compounds [10,11], but enzymatic activity could not be recovered in cell-free extracts [15].
• The cytochrome P450 (P450mor) and its proposed redox partner, a Fe3S4 ferredoxin (Fdmor), were purified for the first time from Mycobacterium sp. strain HE5 [15].
• Nucleotide sequence determination of the encoding operon revealed also the gene encoding the specific reductase, which is required for activity of the P450mor system (B. Sielaff & J. R. Andreesen, unpublished data).
• Thus, the P450mor mono-oxygenase is a typical bacterial P450 system [16], composed of three components: NADH-oxidizing ferredoxin reductase (FdRmor), ferredoxin (Fdmor) as an electron-transfer protein and P450mor, which acts as a mono-oxygenase.
• FdRmor has already been cloned, purified and characterized as a NADH-dependent, FAD-containing protein and shown to be structurally distinct from previously purified P450 reductases (B. Sielaff & J. R. Andreesen, unpublished data), the latter of which all belong to the glutathione reductase-like family.
• An activity of just the cytochrome P450 component has recently been shown for the seemingly identical, recombinant CYP151A2 from Mycobacterium sp. strain RP1 using a heterologous system with both NADPH-dependent ferredoxin reductase and ferredoxin from spinach [17].
• In most reports on bacterial P450 cytochromes activity has been reconstituted with heterologous redox partners [5,9,18-21].
• For biotechnological purposes, strong oxidants like hydrogen peroxide have been used in a few cases for direct involvement of the P450 [22].
• However, less attention has been paid, to date, to the homologous redox partners of P450s.
• The aim of this study was to start a detailed examination of a complete bacterial P450 system distinct from other purified bacterial P450 systems which either utilize a Fe2S2 ferredoxin-like P450cam [23] or belong to the microsomal type of P450s like P450BM3 [24] and are reduced by a diflavin reductase.
• This is the first report on the heterologous expression and purification of all components of a P450 system from an actinobacterium.
• Kinetic investigations were performed on the redox couple FdRmor/Fdmor and morpholine-converting activity could be demonstrated for the reconstituted, homologous P450mor mono-oxygenase.
• Results
• Production and purification of Fdmor variants
• morB, encoding Fdmor, was expressed in Escherichia coli Rosetta(DE3)pLysS as wild-type protein wt-Fdmor, as N-terminal His-tag fusion protein NHis-Fdmor and as C-terminal His-tag fusion protein CHis-Fdmor.
• All proteins were soluble and no inclusion bodies were formed as confirmed by SDS/PAGE analysis.
• The ferredoxins were purified as described in Experimental procedures.
• In the SDS gel (Fig. 1), the purified recombinant proteins appeared larger than expected from their calculated masses, which was similar to findings for the wild-type protein Fdmor isolated from Mycobacterium sp. strain HE5 [15].
• However, the molecular masses determined by MS were in good agreement with those predicted from the sequences (Table 1).
• Absorption spectra were the same for all three recombinant proteins, containing only a single peak at 412 nm, and the protein peak at 280 nm.
• This is a typical feature of Fe3S4 proteins [25] and was found also for wild-type Fdmor isolated from Mycobacterium sp. strain HE5 [15].
• The obtained ratios of the absorbance of the Fe3S4 cluster to the protein-specific absorbance (A280/A412) differed between the recombinant proteins (Table 1).
• The lowest ratio was found for CHis-Fdmor, indicating a high Fe3S4 cluster content.
• Higher ratios were found for NHis-Fdmor and wt-Fdmor, suggesting that the Fe3S4 cluster was not incorporated into these proteins to the same extent.
• In the case of wt-Fdmor, this could be attributed to the different purification protocol, which might have led to some loss of cofactor.
• The highest ratio was found for NHis-Fdmor, which might indicate less efficient incorporation of the Fe3S4 cluster and/or lower stability of the cofactor, compared with CHis-Fdmor and wt-Fdmor.
• EPR-spectroscopy of oxidized wt-Fdmor revealed a single signal with an average g-value of 2.01 which is characteristic of [3Fe-4S]+, S = 1/2 oxidized three-iron cluster (Fig. 2).
• After recording spectra of different Fdmor variants and determining the iron content of these Fdmor solutions by atom absorption spectroscopy, an absorption coefficient for Fdmor of ε412 = 9.8 mm⁻¹·cm⁻¹ could be calculated.
• The amount of purified recombinant ferredoxin was estimated using this absorption coefficient.
• The highest amount was obtained for CHis-Fdmor, whereas wt-Fdmor gave the lowest amount (Table 1), which might again be attributed to the purification procedure.
• Catalytic properties of the recombinant FdRmor/Fdmor couple
• Fdmor was able to stimulate the NADH-dependent reduction of cytochrome c by FdRmor approximately fivefold (B. Sielaff & J. R. Andreesen, unpublished data).
• Screening for other suitable electron acceptors revealed that the further addition of Fdmor enabled reduction of nitroblue tetrazolium (NBT) by FdRmor.
• There was an absolute requirement for Fdmor, as no reduction was observed with NADH and FdRmor alone.
• The influence of the pH on the NADH-dependent reduction of NBT by the FdRmor/Fdmor couple was examined with wt-Fdmor and revealed an optimum at ≈ pH 8.8 (Fig. 3).
• It has been shown previously that the activity of FdRmor is dependent on the type of buffer used (B. Sielaff & J. R. Andreesen, unpublished data).
• In order to exclude this influence, measurements for the determination of the pH optimum were carried out in buffers composed of both 25 mm Tris and 25 mm glycine.
• Potassium chloride had an inhibitory effect on the NBT reducing activity of the FdRmor/Fdmor couple.
• The activity decreased more sharply if up to 50 mm potassium chloride was present.
• This inhibition declined between 50 and 800 mm potassium chloride, where ≈ 50% of the starting activity was reached (Fig. 4).
• Similar results were obtained when sodium chloride was added to the activity assays (data not shown).
• The ferricyanide-reducing activity of FdRmor was not sensitive to ionic strength (data not shown), suggesting that the observed decrease in activity of the FdRmor/Fdmor couple was not caused by an inhibition of the FdRmor activity.
• Steady-state kinetic parameters of FdRmor for wt-Fdmor were determined at pH 8.6 with saturating concentrations of NADH (200 μμ).
• With saturating concentrations of cytochrome c (150 µm), a Michaelis-Menten curve was obtained for the stimulation of the activity of FdRmor towards cytochrome c by wt-Fdmor, indicating an apparent Vmax of 1534 ± 29 electrons min⁻¹ and an apparent Km of FdRmor for wt-Fdmor of 316 ± 17 nm.
• Using NBT (200 µm) as the electron acceptor, an approximately twofold lower Vmax was obtained.
• Owing to a much lower Km value of wt-Fdmor (Table 2), ≈ 60-fold with respect to the Km measured with cytochrome c, the efficiency (Vmax/Km) of wt-Fdmor mediated NBT reduction was ≈ 30-fold higher compared with cytochrome c reduction (Vmax/Km = 4.8_electrons.min⁻¹·nm⁻¹).
• Thus, the FdRmor/Fdmor couple seemed to show a preference for the two-electron acceptor NBT over the one-electron acceptor cytochrome c.
• In order to check whether the added sequence providing the His-tag to the recombinant ferredoxins had an influence on the activity of the FdRmor/Fdmor couple, kinetic parameters were determined with NHis-Fdmor and CHis-Fdmor.
• Using cytochrome c as the electron acceptor, activities with a saturating concentration of NHis-Fdmor or CHis-Fdmor could not be determined correctly, as these recombinant ferredoxins showed unspecific activities with NADH and cytochrome c without any addition of FdRmor.
• These background activities were negligible at low ferredoxin concentrations, but measurements at apparent saturating concentrations of ferredoxin yielded such high activities that it was not possible to measure initial velocities over a reasonable period.
• Thus, Km and Vmax values could not be determined under these conditions.
• However, from the slope of the initial linear range of the kinetic plot, the constants Vmax/Km of 1.1 electrons.min⁻¹·nM⁻¹ for NHis-Fdmor and_Vmax/Km_of 0.9 electrons.min⁻¹·nm⁻¹ for CHis-Fdmor could be estimated as approximate figure.
• These were approximately fivefold lower than the Vmax/Km determined with wt-Fdmor.
• NHis-Fdmor and CHis-Fdmor showed reducing activities towards NBT, similar to those seen in cytochrome c assays.
• In comparison with cytochrome c activities, there was a lower reduction of NBT by the FdRmor/Fdmor couple as well as by His-tagged Fdmor on its own.
• Therefore, initial velocities could be measured with saturating concentrations of ferredoxin.
• However, kinetic plots did not show a typical Michaelis-Menten curve.
• Instead of reaching a plateau, velocities continued to increase in a linear dependence on the ferredoxin concentration (Fig. 5), which could be attributed to the unspecific background activities of His-tagged ferredoxins.
• Therefore, the data were fitted to a modified Michaelis-Menten equation (Experimental procedures) where a linear term was added to describe the FdRmor-independent NBT reduction by the ferredoxin.
• This method revealed the kinetic parameters of FdRmor for NHis-Fdmor or CHis-Fdmor, which were found to be in the same range as those determined for wt-Fdmor (Table 2).
• Production and purification of recombinant P450mor
• morA, encoding P450mor, was expressed as fusion protein with an N-terminal His-tag in E. coli Rosetta(DE3)pLysS cells.
• The reduced CO difference spectra of cytosolic extracts showed a characteristic maximum absorbance peak at 450 nm.
• Supplementation of the growth medium with the heme precursor d-aminolevulinic acid increased the expression level of P450mor up to fivefold, suggesting that heme was limiting during the heterologous expression conditions.
• SDS/PAGE analysis revealed that apparently no inclusion bodies were formed.
• The protein was isolated by a single chromatography step on a Ni2+ affinity column and was judged to be homogenous by SDS/PAGE analysis.
• NHis-P450mor showed a molecular mass of 46 000 Da in SDS/PAGE, appearing larger than the wild-type P450mor (Fig. 1), as expected as a result from the added sequence.
• MS revealed a molecular mass of 46 705 Da which was in good agreement with the calculated mass of 46 700 Da for NHis-P450mor.
• The UV-Vis spectrum of NHis-P450mor was identical to that of wild-type P450mor, isolated previously from