Cross-linking of dicyclotyrosine by the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis proceeds through a catalytic shunt pathway
2017; Elsevier BV; Volume: 292; Issue: 33 Linguagem: Inglês
10.1074/jbc.m117.794099
ISSN1083-351X
AutoresKednerlin Dornevil, Ian Davis, Andrew J. Fielding, James R. Terrell, Li Ma, Aimin Liu,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoCYP121, the cytochrome P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C–C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C–C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme–substrate complex. This intermediate exhibits a high-spin (S = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical–based mechanisms for the C–C bond coupling reaction. CYP121, the cytochrome P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C–C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C–C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme–substrate complex. This intermediate exhibits a high-spin (S = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical–based mechanisms for the C–C bond coupling reaction. Mycobacterium tuberculosis causes more deaths annually worldwide than any other known pathogen. As the causative agent of tuberculosis in humans, it is one of the most dangerous and difficult-to-combat bacterial infections. Approximately 10.4 million people suffered from tuberculosis in 2015 with 1.5 million deaths (1.World Health Organization Global Tuberculosis Report 2016. World Health Organization, Geneva, Switzerland2016Google Scholar). A primary reason for the effectiveness of the pathogen is the recent development of drug- and multidrug-resistant M. tuberculosis strains. Nearly 10% of new infection cases are multidrug-resistant tuberculosis. Resistance to common antibiotics makes treatment very difficult. As the number of strains resistant to frontline drugs grows, pressure is increasing for the identification of potential new targets to combat M. tuberculosis infections and the development of new types of drugs and drug classes (2.Matsumoto M. Hashizume H. Tsubouchi H. Sasaki H. Itotani M. Kuroda H. Tomishige T. Kawasaki M. Komatsu M. 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The gene encoding CYP121 in M. tuberculosis was found in an operon-like structure with the gene rv2275 (3.Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry 3rd, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. et al.Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.Nature. 1998; 393: 537-544Crossref PubMed Scopus (6512) Google Scholar, 17.Roback P. Beard J. Baumann D. Gille C. Henry K. Krohn S. Wiste H. Voskuil M.I. Rainville C. Rutherford R. A predicted operon map for Mycobacterium tuberculosis.Nucleic Acids Res. 2007; 35: 5085-5095Crossref PubMed Scopus (64) Google Scholar). Characterization of Rv2275 in Escherichia coli revealed that the products were mainly tyrosine-containing cyclo dipeptides, the majority of which were cyclo-(l-Tyr-l-Tyr) (cYY) 3The abbreviations used are: cYY, dicyclotyrosine; Ac-O-O-Fe(III), acetate-O-O-ferric heme; P450, cytochrome P450; PAA, peracetic acid; ABTS, 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonate); PhIO, cYY iodosylbenzene. 3The abbreviations used are: cYY, dicyclotyrosine; Ac-O-O-Fe(III), acetate-O-O-ferric heme; P450, cytochrome P450; PAA, peracetic acid; ABTS, 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonate); PhIO, cYY iodosylbenzene. (18.Gondry M. Sauguet L. Belin P. Thai R. Amouroux R. Tellier C. Tuphile K. Jacquet M. Braud S. Courçon M. Masson C. Dubois S. Lautru S. Lecoq A. Hashimoto S. Genet R. Pernodet J.L. Cyclodipeptide synthases are a family of tRNA-dependent peptide bond–forming enzymes.Nat. Chem. Biol. 2009; 5: 414-420Crossref PubMed Scopus (179) Google Scholar). At the same time, the crystal structure for CYP121 in complex with cYY was also solved by Belin et al. (19.Belin P. Le Du M.H. Fielding A. Lequin O. Jacquet M. Charbonnier J.B. Lecoq A. Thai R. Courçon M. Masson C. Dugave C. Genet R. Pernodet J.L. Gondry M. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7426-7431Crossref PubMed Scopus (154) Google Scholar). Assays conducted on CYP121 utilizing a ferredoxin and ferredoxin reductase system demonstrated that CYP121 catalyzes multiple turnovers of cYY to form mycocyclosin as the single major product in the presence of NADPH (19.Belin P. Le Du M.H. Fielding A. Lequin O. Jacquet M. Charbonnier J.B. Lecoq A. Thai R. Courçon M. Masson C. Dugave C. Genet R. Pernodet J.L. Gondry M. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7426-7431Crossref PubMed Scopus (154) Google Scholar). The product exhibits a cross-link between the respective carbons in the ortho position of cYY tyrosine moieties. P450 enzymes are normally known to promote a wide range of catalytic activities of aliphatic and aromatic hydroxylation, dealkylation, desaturation, epoxidation, deamination, dehalogenation, dehydration, and isomerization. The C–C bond formation represents an unusual activity of the P450 enzyme superfamily (20.Mizutani M. Sato F. Unusual P450 reactions in plant secondary metabolism.Arch. Biochem. Biophys. 2011; 507: 194-203Crossref PubMed Scopus (129) Google Scholar, 21.Giessen T.W. Marahiel M.A. Rational and combinatorial tailoring of bioactive cyclic dipeptides.Front. Microbiol. 2015; 6: 785Crossref PubMed Scopus (48) Google Scholar). However, important chemical and biological questions remain unanswered regarding CYP121. The mechanism for cross-link formation and the identity of the oxidizing species or the physiological relevance of mycocyclosin are still unclear. A quantum mechanics/molecular mechanics study supports the catalytic mechanism via formation of a diradical intermediate species with the cross-link being formed non-enzymatically in solution (22.Dumas V.G. Defelipe L.A. Petruk A.A. Turjanski A.G. Marti M.A. QM/MM study of the C—C coupling reaction mechanism of CYP121, an essential cytochrome P450 of Mycobacterium tuberculosis.Proteins. 2014; 82: 1004-1021Crossref PubMed Scopus (25) Google Scholar). The current work expands on previous research by investigating the reaction pathway of the CYP121 system. CYP121 is uniquely attractive because of the non-canonical P450 chemistry it catalyzes and the question of whether or not it follows the classical mechanism of P450s. The mechanistic question under investigation includes the “short circuit,” also known as the catalytic “peroxide shunt” pathway, for the formation of a ferric hydroperoxide adduct complex and the subsequent oxo-ferryl species (Fig. 1) (23.Porter T.D. Coon M.J. Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms.J. Biol. Chem. 1991; 266: 13469-13472Abstract Full Text PDF PubMed Google Scholar, 24.Sono M. Roach M.P. Coulter E.D. Dawson J.H. Heme-containing oxygenases.Chem. Rev. 1996; 96: 2841-2888Crossref PubMed Scopus (2107) Google Scholar). Toward this aim, we have carried out rapid kinetics, spectroscopy, and LC-MS analysis to investigate the CYP121 reaction mechanism. The as-isolated CYP121 exhibits a Soret peak centered at 416 nm. Two absorbance features at 538 and 565 nm in the α/β region and an additional minor band at 648 nm are also present. CYP121 displays type I characteristic spectral changes in the UV-visible heme Soret spectrum upon binding of cYY (Fig. 2A), as is frequently observed in P450 enzymes during the binding of endogenous substrates and xenobiotics (25.Isin E.M. Guengerich F.P. Substrate binding to cytochromes P450.Anal. Bioanal. Chem. 2008; 392: 1019-1030Crossref PubMed Scopus (93) Google Scholar). After substrate binding, the Soret peak blue-shifts to 395 nm. However, a significant shoulder peak remains even when saturating concentrations of cYY (400 μm, 80:1 ratio of the substrate over enzyme, 20 times the cYY Kd value of 21.3 μm) (19.Belin P. Le Du M.H. Fielding A. Lequin O. Jacquet M. Charbonnier J.B. Lecoq A. Thai R. Courçon M. Masson C. Dugave C. Genet R. Pernodet J.L. Gondry M. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7426-7431Crossref PubMed Scopus (154) Google Scholar) were used. This phenomenon has previously been observed among the P450 family as well as in CYP121 (19.Belin P. Le Du M.H. Fielding A. Lequin O. Jacquet M. Charbonnier J.B. Lecoq A. Thai R. Courçon M. Masson C. Dugave C. Genet R. Pernodet J.L. Gondry M. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7426-7431Crossref PubMed Scopus (154) Google Scholar). In the α/β region, additional changes occurred with the loss of the two peaks to generate a new broad feature at 516 nm with shoulders at 541 and 571 nm, whereas the charge transfer peak at 651 nm increased in intensity (19.Belin P. Le Du M.H. Fielding A. Lequin O. Jacquet M. Charbonnier J.B. Lecoq A. Thai R. Courçon M. Masson C. Dugave C. Genet R. Pernodet J.L. Gondry M. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7426-7431Crossref PubMed Scopus (154) Google Scholar). The spectroscopic changes observed for substrate binding are summarized in Table 1. The spin transition was less pronounced when monitored by low-temperature EPR spectroscopy for samples frozen by liquid ethane. The incomplete spin-state conversion most likely originates from the low temperature used in the EPR studies. A slight decrease in the g-anisotropy of the low-spin species and a new high-spin resonance at g = 8 is observed (Fig. 2B).Table 1Summary of spectra profile changes observed for substrate binding to CYP121 and ES complex reaction with peracetic acid as monitored by stopped-flow UV-visible spectroscopyPositive peakNegative peakIsosbestic pointTransition123123123456nmnmnmcYY binding393508651423575410460540597674ES + PA433573393517651345404473551629672 Open table in a new tab Stopped-flow experiments to determine the microscopic rate constants (kon and koff) for cYY binding to Fe(III)-CYP121 were performed by monitoring the formation of the ES complex at 388 nm using a stopped-flow spectrometer. This wavelength was chosen because in the difference spectra, the largest amplitude change is at 388 nm (Fig. 2C). Fig. 3A shows stopped-flow time traces varying the [cYY] from 20 to 700 μm. Fitting the kinetic traces to single exponential equations resulted in a very poor fit with significant residual amplitudes that show systematic dependence with [cYY]. In contrast, a very good fit was obtained by using a two-exponential equation (Fig. 3A). The plots of the observed rates (1/τ1 and 1/τ2), from double-exponential fitting of the [cYY] dependence are shown in Fig. 3B. Whereas 1/τ1 shows linear [cYY] dependence, 1/τ2 shows parabolic concentration dependence, suggesting a stepwise mechanism for cYY binding (Reaction 1) to form the binary ES complex. Fitting both 1/τ1 to a linear equation and 1/τ2 to a hyperbolic equation yielded non-zero y intercepts, suggesting that both steps are reversible. From the replotting of the [cYY] dependence data (Fig. 3C), taking both the sum (1/τ1 + 1/τ2) and the product (1/τ1 × 1/τ2) of the observed reciprocal relaxation times, the microscopic rate constants (k1, k−1, k2, and k−2) can be calculated from the slope and the y intercept from the two graphs (Reaction 1) (26.Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-state Enzyme Systems. Wiley-Interscience, New York1993Google Scholar). The second-order rate constant (k1) of 0.065 ± 0.002 μm−1 s−1 is fairly low where the initial binding step is slower than both k−1 and k2 (k1 < k−1 and k2 when [cYY] < 62 μm). The reverse rates (k−1 and k−2) are also on the same order of magnitude and have similar values to k2. These suggest a highly reversible system where ES* and ES are in rapid equilibrium. E+S⇌k−1=4.2±0.6s−1k1=0.065±0.02μM−1s−1ES*⇌k−1=3.2±0.9s−1k2=4.0±1.2s−1ESReaction1(Eq. 1) Reaction 1 shows the proposed multistep mechanism for cYY binding to Fe(III)CYP121, with rate constants obtained from double exponential fitting of stopped-flow data from [cYY] dependence data (Fig. 3). If CYP121 follows the general cytochrome P450 mechanism, it would be able to generate the enzyme-based key oxidant, compound I, through the well-established peroxide shunt pathway. The peroxide shunt pathway bypasses the need for NAD(P)H and a redox mediator system to supply electrons and protons to the heme-bound O2 (27.Nordblom G.D. White R.E. Coon M.J. Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome-P-450.Arch. Biochem. Biophys. 1976; 175: 524-533Crossref PubMed Scopus (288) Google Scholar, 28.Gustafsson J.A. Rondahl L. Bergman J. Iodosylbenzene derivatives as oxygen donors in cytochrome P-450 catalyzed steroid hydroxylations.Biochemistry. 1979; 18: 865-870Crossref PubMed Scopus (63) Google Scholar, 29.White R.E. Sligar S.G. Coon M.J. Evidence for a homolytic mechanism of peroxide oxygen-oxygen bond cleavage during substrate hydroxylation by cytochrome P-450.J. Biol. Chem. 1980; 255: 11108-11111Abstract Full Text PDF PubMed Google Scholar). Among H2O2, cumene peroxide, t-butyl peroxide, meta-chloroperoxybenzoic acid, and peracetic acid (PAA) tried at pH 7.4, only H2O2 and PAA gave significant reactions. Because PAA has the most apparent reaction at lower concentrations during our initial tests, the following work described in this study was mostly focused on PAA. The reaction was initiated by incubating the peroxide oxidant with 5 μm CYP121 premixed with cYY to determine whether the shunt pathway would be a viable route to generate the C–C cross-linked product. A parallel experiment was performed in the absence of substrate as a control to assist the identification of the intermediate species. The enzyme was first preincubated with the substrate (600 μm) and subsequently mixed with increasing concentrations of PAA, and the reaction was monitored for 30 s by stopped-flow UV-visible spectroscopy (Fig. 4A). Over the course of the reaction, the difference spectra show several transitions (Fig. 4B). During the first 300 ms, the ES complex absorbance at 395 nm decreases concomitantly with an increase at 427 nm. As the reaction continues, the 395-nm Soret peak of the ES complex continues to decrease while the 427 nm peak increases and red-shifts to 433 nm. Finally, when the reaction proceeds for longer than 10 s, the isosbestic points become less clear, suggesting that heme bleaching becomes a contributing factor in the reaction. Monitoring the reaction rate as a function of PAA concentration allows for the determination of the apparent pseudo-first-order rate constant kobs of (7.2 ± 0.1) × 10−4 s−1. For comparison, when the enzyme is reacted with PAA in the absence of substrate, the first 100 ms generate a new intermediate species (Fig. 5A). The formation of the intermediate species is described by a decrease in the intensity of the Soret peak with an increase in the shoulder peak near 379 nm. Near the α/β region of the spectra, decreases at 543 and 574 nm are observed with the formation of two minor peaks at 616 and 704 nm. The difference spectra clearly show the formation of a new species. Five isosbestic points at 300, 392, 447, 517, and 584 nm are also observed in the difference spectra (Fig. 5A, inset). The clear isosbestic points suggest a direct transition from resting state heme directly to the first intermediate species. From 100 ms to ∼2 s, a second transition occurs, forming a second intermediate species (Fig. 5B). This second intermediate is distinct with isosbestic points at 296, 340, 427, and 580 nm. The second intermediate is highlighted by two noticeable absorption changes at 421 and 440 nm, decreasing and increasing, respectively. The final transition occurs from 2 to 20 s of CYP121 reacting with PAA (Fig. 5C). The difference spectra contain two prominent peaks centered at 412 nm (increasing) and 440 nm (decreasing) (Fig. 5C, inset). A complete profile of the transitions observed is compiled in Table 2. The 433-nm species found in the reaction of ES complex with PAA is not present in the reaction when cYY is absent.Table 2Summary of intermediate spectrum profilesPositive peakNegative peakIsosbestic pointTransition1234512341234nmnmnm100 ms3794696167044215435743133926012 s31838244053557135942158729634042758020 s412495595624681374440538572393425583 Open table in a new tab Because the P450 enzyme-mediated C–C bond coupling mechanism has not yet been elucidated, one cannot assume that the shunt pathway in CYP121 will lead to the generation of the reported product. To demonstrate the relevance of PAA as an oxidative source for mechanistic studies, the reaction mixture was characterized by LC-MS. When the substrate was analyzed alone or pairwise in the presence of either PAA or CYP121 only, a single peak elutes with a retention time of ∼8 min (Fig. 6A). This peak possesses an m/z of 325, which is the expected value for the cYY substrate (Fig. 6B). When all three components, CYP121, cYY, and PAA, are combined and allowed to react (see “Experimental procedures”), the reaction mixture contains new peaks, and the peak with a retention time close to 5 min shows an m/z of 323, which is consistent with the cross-linked mycocyclosin product (Fig. 6, C and D). These data demonstrate that CYP121 can utilize the shunt pathway to carry out the C–C cross-linking reaction on cYY and generate mycocyclosin. To gain more insight into the CYP121 cross-linking reaction, rapid freeze-quench EPR samples were made in which CYP121 was rapidly mixed with PAA in either the presence or absence of cYY before quenching in liquid ethane at various time points. The quenching times chosen were guided by our stopped-flow studies described above. When the reaction of the ES complex with PAA is quenched at 5 ms, EPR data reveal a nearly complete disappearance of the ES complex EPR signal (≥75%). Instead, the EPR spectra from samples trapped at different time points in the millisecond time window show a new high-spin (S = 5/2) ferric heme species with g values of 6.87, 5.77, and 2.00 (Fig. 7). When allowed to react for longer times, the high-spin species decreases in intensity (Table 3). However, the decay of the high-spin species was not accompanied by regeneration of the low-spin ferric signal. Noticeably, a new EPR-silent heme species was formed during this time. The remaining minor low-spin heme signal also continuously decays during this period. During the same time window, the 427-nm species was developed and shifted to 433 nm in the stopped-flow experiments. The final sample quenched at 10 s after mixing gives a spectrum with no sign of the low-spin heme. The high-spin ferric signal intensity is significantly reduced compared with the 5 ms sample. A new significant portion of adventitious iron, presumably from heme degradation, and a free radical species are observed. Minor EPR species at g = 2 are observed in the samples, but they are unlike any characterized tyrosyl radicals. Thus, the radical species are unlikely to be associated with the cYY reaction but are more likely to be intermediates of side reactions toward heme degradation. When the rapid freeze-quench EPR study was performed with 0.4 mm PAA with more time points in the first 180 s, similar results were obtained (i.e. the formation of the g = 6 species maximizes and then later starts to decay). The only difference is that the g = 6 species is lower in intensity compared with the 10 mm PAA, and the heme-bleaching g = 4.3 signal does not occur at the end of the reaction.Table 3Summary of the resonance components in the rapid freeze-quench EPR spectra of ES complex reacting with PAAg valueES complex5 ms160 ms300 ms10 s%%%%%5.77010098.980366.870100778008.111001001001000 Open table in a new tab For the reaction of CYP121 alone with PAA, a series of freeze-quench samples were prepared and analyzed by EPR (Fig. 8). After quenching at 100 ms, a significant decrease in the low-spin heme signal was observed with the formation of a minor new high-spin species with g values of 6.67, 5.77, and 2.0. This species is distinctly different from the high-spin heme intermediate observed in the reaction of the ES complex with PAA. After reacting for longer times, the 2-s sample resulted in a further reduction of the low-spin heme and a continued increase of the high-spin
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