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reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group Vito Barracco a, d, 1, Roberta Moschini a, c, 1, Giovanni Renzone b, Mario Cappiello a, c, Francesco Balestri a, c, Andrea Scaloni b, Umberto Mura a, Antonella Del-Corso a, c, * a
University of Pisa, Department of Biology, Biochemistry Unit, Via S. Zeno, 51, Pisa, Italy Proteomics & Mass Spectrometry Laboratory, ISPAAM-CNR, Via Argine, 1085, Napoli, Italy Interdepartmental Research Center Nutrafood ‘‘Nutraceuticals and Food for Health’‘, University of Pisa, Pisa, Italy d PhD Student at the Tuscany Region “Pegaso” PhD School in Biochemistry and Molecular Biology, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2019 Accepted 14 November 2019 Available online xxx
Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed. © 2019 Elsevier Inc. All rights reserved.
Keywords: Carbonyl reductase 1 3-Glutathionyl-4-hydroxynonanal disproportionation NADP(H) prosthetic group
1. Introduction Carbonyl reductase 1 (CBR1), a cytosolic monomeric NADPþdependent secondary alcohol dehydrogenase [E.C.1.1.1.184] is common in mammals [1] and found in several human tissues [2]. The reductase activity of the enzyme is exerted in a variety of substrates, such as eicosanoids, steroids, lipid peroxidation-derived carbonyl compounds, as well as orto- or para-xenobiotic quinone derivatives [3e5]. The active site of CBR1 is quite complex. CBR1 contains a very flexible wide interactive region, which tightly closes around the substrate after binding [6]. A glutathione-binding site, close by the active site, has also been identified [7,8]. This is in
Abbreviations: CBR1, carbonyl reductase 1; DTT, D,L-dithiothreitol; GSDHN, 3glutathionyl-1,4-dihydroxynonane; GSH, reduced glutathione; GSHNA-lactone, 3glutathionyl-4-hydroxynonanoic-g-lactone; GSHNE, 3glutathionyl-4hydroxynonal; GSNA, 3-glutathionyl-nonanal; hCBR1, human recombinant CBR1; HNE, 4-hydroxy-2-nonenal. * Corresponding author. Department of Biology, Biochemistry Unit, via S. Zeno 51, 56127, Pisa, Italy. E-mail address: [email protected] (A. Del-Corso). 1 These authors contributed equally to the work.
addition to the expected interplay of functional residues at the active site of CBR1, such as Cys227 which in its negative thiolate status, orients the substrate [9,10] and Trp229, Ala235 and Met241 involved in the binding of the hydrophobic moieties of substrates [5]. The ability of CBR1 to intervene as a reductase not only on keto groups, as expected for a secondary alcohol dehydrogenase, but also on aldehydic groups, is still an open issue. Although aldehydes (including 4-hydroxy-2-nonenal - HNE) are poor substrates for the enzyme [5], a 10% reduction of the aldehydic group, together with an oxo-reduction, has been reported when 4-oxo-trans-2-nonenal was used as a substrate [11]. CBR1 appeared to be much more efficient in catalyzing the reduction of glutationylated aldehydes [12], and this evidence is conceivably supported by the abovementioned GSH binding site. The oxo group in position 9 of prostaglandin-A1 used as substrate for the enzyme, was reduced only on the glutathionylated adduct. The latter is also able to inhibit the 15-hydroxyprostaglandin dehydrogenase activity of CBR1 on prostaglandin-B1 [7,10]. The strong effect of the glutathionyl moiety of substrates in addressing the substrate on the active site makes the effect of
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Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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Cys227 any more critical. However, this did not apply to S-nitrosoglutathione, whose efficient reduction by the enzyme is subjected to Cys227 control [7,13]. All this evidence underlines the complexity of the substrate allocation at the active site of CBR1. The ability of CBR1 to intervene on 3-glutathionyl-4hydroxynonanal (GSHNE) metabolism both as a dehydrogenase, on the hemiacetal hydroxyl group [14], and as a reductase, on the free aldehydic carbonyl group [12], catalyses an overall disproportionation reaction of the molecule at sub-stoichiometric levels of the pyridine cofactor [15]. Thus, sustained by a NADPþ/NADPH recycle, GSHNE was both oxidized to the corresponding 3glutathionyl-4-hydroxynonanoic-g-lactone (GSHNA-lactone), ready for cell extrusion [16] and reduced to 3-glutathionyl-1,4dihydroxynonane (GSDHN), a pro-inflammatory molecular signal [17]. This paper highlights that CBR1 may act as catalyst of the disproportionation reaction without any cofactor exchange with the solution, being able of a redox recycle of a stoichiometric amount of bound cofactor.
2.4. Liquid chromatography-mass spectrometry analysis of GSHNE reaction products Assay mixtures containing GSHNE (prepared as described in Section 2.5) were incubated in different conditions either in the absence or in the presence hCBR1, and analysed in order to determine the reaction extent on a time-course basis. Identical aliquots sampled at different times were immediately frozen, lyophilized and then stored at 80 C, until further use. Samples were dissolved in 0.1% trifluoroacetic acid and subjected to nanoLC-ESI-LIT-MS/MS analysis with an LTQ XL mass spectrometer (ThermoFisher, USA) equipped with a Proxeon nanospray source, which was connected to an UltiMate 3000 RSLC nano-liquid chromatographer (ThermoFisher, USA) [14,15]. Analyte mixtures were resolved, and the corresponding spectra were acquired [14,15]. Assignment of MS/MS spectra to specific glutathione derivatives and semi-quantitative measurement of the reaction products were then carried out [14,15]. 2.5. Preparation of glutathionylated substrates
2. Materials and methods 2.1. Materials All reagents, solvents and filtration devices used were as previously described [14].
GSHNE and 3-glutathionylnonanal (GSNA) were prepared by incubating 1.5 mM GSH in 50 mM sodium phosphate buffer, pH 7.4, with 1 mM of either HNE or trans-2-nonenal, respectively [14]. Taking into account the disappearance of HNE and trans-2-nonenal (by measurements at 224 nm), the residual GSH [18] and the spontaneous oxidation of GSH [19], a stoichiometric consumption of GSH and HNE was observed, with less than 10% residual alkenals. Both thiolated adducts were stored at 80 C until used.
2.2. Enzyme assay, expression and purification
2.6. Other methods
The activity of human recombinant carbonyl reductase 1 (hCBR1) was determined at 37 C in an assay mixture containing, in 50 mM sodium phosphate buffer pH 8.4, 0.18 mM NADPþ and 0.1 mM GSHNE as substrate [12]. The reaction was monitored following the increase in absorbance at 340 nm linked to the reduction of NADPþ. One Unit of enzyme activity is the amount of enzyme that catalyses the conversion of 1 mmol of substrate/min in the specified assay conditions. hCBR1 was expressed and purified to electrophoretic homogeneity as previously described [12]. Purified hCBR1 samples, at a concentration of 0.15 mg/mL (5 mM, calculated on the basis of a molecular mass of 31 KDa) and displaying a specific activity of 50 U/ mg, were stored at 80 C in a 10 mM sodium phosphate buffer pH 7.0 (Standard Buffer, SB) containing 1 mM D,L-dithiothreitol (DTT), 0.05 mM NADPþ, 0.75 M NaCl and 33% w/v glycerol. Before use, the enzyme was extensively dialyzed against SB.
Protein concentration was determined according to the Bradford method [20]. Fluorescence spectra were obtained with an FP-6500 spectrofluorometer (Jasco, Easton, MD, USA) using an excitation wavelength of 345 nm. Emission fluorescence was measured at wavelengths of between 400 and 600 nm.
2.3. Dialysis protocols In order to obtain a NADP-free hCBR1, 3 mM of purified hCBR1 was dialyzed at 4 C through ultra-membrane filters (10 kDa cutoff) against 2 103 vol of SB supplemented with 1.5 M NaCl. A second dialysis step against 4 103 vol of SB in the above conditions was then carried out. The enzyme concentration appeared critical for the recovery of the enzyme activity after dialysis. In order to ensure a recovery higher than 80%, both in terms of protein concentration and specific activity, the protein concentration was never allowed to be lower than 0.6 mM throughout the dialysis. NADP-free hCBR1 (3 mM) was loaded with NADPþ or NADPH by incubation for 15 min at 25 C in the presence of 30 mM of either NADPþ or NADPH, respectively, followed by dialysis against 4 103 vol of SB.
3. Results and discussion When a highly-purified preparation of hCBR1 (63 nM) extensively dialyzed against 4 103 vol of SB (leading to a residual nominal cofactor level in the solution of 0.16 nM, i.e. less than 0.25% with respect to the enzyme concentration) was added to a GSHNE solution, both the oxidation and reduction of the adduct occurred, although the cofactor was nominally absent in the assay. As shown in Fig. 1A, the disappearance of the substrate and the parallel generation of both GSHNA-lactone and GSDHN occurred at a detectable rate, halving the substrate concentration within 20 min. No significant differences were observed in the reaction rate when the above-described solution was supplemented by NADPþ up to a nominal concentration of the cofactor of 63 nM, accounting for a cofactor/enzyme molar ratio of 1 (data not shown). This could be due to either a disproportionation reaction of GSHNE occurring in the absence of the cofactor, or alternatively, to the fact that, despite the very extensive dialysis step, the cofactor was firmly retained by the enzyme and recycled on it while performing catalysis. To the best of our knowledge, no structural evidence is available to support the ability of the enzyme to bind two substrate molecules at the active site leading to their disproportionation reaction. The involvement of the cofactor trapped on the active site, while catalysis is taking place, thus appeared to be the most likely explanation. Indeed, evidence of NADP bound on hCBR1 following purification has already been reported [21]. The ability of pyridine cofactors to tightly bind enzymes is not infrequent [22,23], and the
Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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Fig. 1. Time-course of GSHNE transformation catalyzed by hCBR1. GSHNE (100 mM) was incubated at 37 C in a sodium phosphate buffer 50 mM pH 6.2 in various conditions in the presence of 94 mU/mL (63 nM) of purified hCBR1 and the reaction mixture analysed by mass spectrometry (see Section 2.4). Plots are reported of the peak areas of the molecular species at m/z values 464.25 (GSHNE - circles), 466.25 (GSDHN - squares) and 462.23 (GSHNA-lactone - triangles), as obtained by integrating the extracted ion currents relative to nanoLC-ESI-MS runs at different incubation times. The experimental points are reported with the corresponding error bars, and represent the average of at least three independent measurements. Panel A: the incubation was performed with the enzyme previously dialyzed against 4 103 vol of SB. Panel B: the incubations were performed with the NADP-free hCBR1 (see Section 2.3). Curves marked with 1, 2 and 3 refer to incubations performed supplementing the solution with zero, 63 nM and 313 nM NADPþ, respectively.
implications of such an event on the functional features of the involved enzymes have been underlined [24]. Such evidence for CBR1 is the rational base to further the apparent disproportionation reaction of GSHNE. Gaining key evidence to support this point entailed assessing presence of the cofactor on hCBR1 for catalysis. The possibility of removing the bound cofactor, whenever present, from the enzyme was further evaluated by adopting more severe dialysis conditions. Thus, the application of the dialysis protocol (see Section 2.3) to hCBR1 led to the complete abolishment of the disproportionation reaction of the substrate (Fig. 1B). The disappearance of the substrate and the parallel formation of GSDHN and GSHNA-lactone occurred only following the addition of low concentrations of the cofactor, accounting for a cofactor/enzyme molar ratio from 1 to 5. Since the GSHNE transformation far exceeded the concentration of the pyridine cofactor, these results confirmed the occurrence of an overall disproportionation reaction, ruling out the potential of any NADP(H)-independent ability of hCBR1 to catalyze this reaction. The efficiency of the bound cofactor in intervening in the redox step was then assessed. This was achieved by exploiting the emission fluorescence spectrum shown by NADPH, but not by NADPþ, with a maximum emission at 460 nm when excited at 345 nm. Following the interaction of the cofactor with the protein [25,26], the NADPH spectrum showed a modest hypsochromic shift and an increase in emission intensity. Fig. 2 shows the differential fluorescence spectrum between the trace recorded in the case of a hCBR1 solution (1.2 mM) saturated with NADPH, and the counterpart of an equally concentrated NADPH solution in the buffer. The inset in Fig. 2 reports the saturation process of hCBR1 by the cofactor as the differential emission intensity at 460 nm, which resulted from the gradual addition of NADPH to the enzyme. No fluorescence emission was measured after the enzyme was saturated with NADPþ (Fig. 2, trace 4). This confirms the effectiveness of this methodological approach to
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Fig. 2. Loading of hCBR1 with NADPH. NADP-free hCBR1 (see Section 2.4) (1.2 mM) in SB was supplemented with increasing concentrations of NADPH. The corresponding fluorescence emission spectra (345 nm excitation wavelength) were recorded. Traces 1 and 2 represent the spectrum of 32 mM NADPH in SB alone or in the presence of 1.2 mM hCBR1, respectively. The dotted line refers to the differential spectrum obtained by subtracting trace 1 from trace 2. Traces 3 and 4 refer to the spectra of the NADP-free hCBR1 before and after the addition of 32 mM NADPþ, respectively. The inset shows the differential emission fluorescence at 450 nm as a function of the cofactor/enzyme ratio obtained upon the addition of exogenous NADPH.
follow the redox status of the cofactor. To verify the involvement of the bound cofactor in the disproportionation reaction, the enzyme deprived of the endogenous cofactor (i.e. NADP-free hCBR1, as defined in 2.3) was loaded with either NADPþ or NADPH. Following an extensive dialysis, it was then subjected to a spectrofluorometric analysis in various conditions thereby allowing the enzyme to exert catalysis. The enzyme loaded with NADPH (as reported in Fig. 2) was thus subjected to extensive dialysis against 4 103 vol of SB, and the corresponding fluorescence spectrum was measured. This spectrum (Fig. 3A, trace 1) coincided with the calculated differential spectrum in Fig. 2, and thus indicated a complete retention of the loaded cofactor after dialysis. The addition of 1.6 mM GSNA to the solution (corresponding to a two-fold concentration with respect to the enzyme) (Fig. 3A, trace 2) led to a very rapid (a few seconds) decrease in fluorescence, possibly associated with the NADPH-dependent reduction of the substrate. Likewise, the in situ reduction of the bound NADPþ was followed by adding GSHNE to hCBR1 pre-loaded with NADPþ (Fig. 3B, traces 1 to 5). A gradual increase in the intensity of the fluorescence was observed a few seconds after each addition of GSHNE, which reached a maximum at a substrate level that was approximately 8fold the enzyme concentration (trace 5). This fluorescence emission represented 65% of the NADPH load capacity of the enzyme (Fig. 3A, trace 1). It was compatible with the in situ reduction of the bound NADPþ by a substrate such as GSHNE, which may undergo both oxidation and reduction reactions. When the solution was supplemented with GSNA at a substrate/ enzyme molar ratio of 2, a very rapid decrease in fluorescence was observed (Fig. 3B, trace 6), which was compatible with NADPH oxidation. Note that over time the intensity of the fluorescence slowly started to recover (Fig. 3B, inset), likely due to presence of residual GSHNE in the mixture. Implementing the GSHNE concentration and thus favouring the oxidative arm of the disproportionation cycle, led to a rapid increase in the emission intensity up to approximately 50% of the initial value (Fig. 3B, trace 5). In order to rule out the possibility of a mobilization of the bound
Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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Fig. 3. Separation of the two arms of the GSHNE disproportionation reaction. Panel A: hCBR1 saturated with NADPH, as described in Fig. 2, was extensively dialysed against 4 103 vol of SB, and the corresponding spectrum was recorded at an enzyme concentration of 0.8 mM (trace 1). Trace 2 refers to the enzyme sample supplemented with 1.6 mM GSNA. Panel B: NADP-free hCBR1 was saturated with NADPþ (see Section 2.3), and the corresponding spectrum was recorded at an enzyme concentration of 0.8 mM (trace 1). Traces 2, 3, 4, 5 refer to the emission spectra recorded after the gradual addition of GSHNE to the enzyme solution to obtain final concentrations of 0.8 mM, 1.6 mM, 3.2 mM and 6.4 mM, respectively. Trace 6 refers to the further addition of 1.6 mM GSNA to the sample of trace 5. The inset shows the slow recovery of fluorescence emission from the sample of trace 6, which was enhanced by again supplementing the solution with 6.4 mM GSHNE at the time indicated by the arrow.
cofactor during catalysis, a dialysis step was inserted between the forward and reverse reactions. This step was designed to remove the excess GSHNE and, if present, the mobilized cofactor. After the in situ reduction of NADPþ by GSHNE (Fig. 4, trace 2), the recovered hCBR1 activity (85% of the initial value) after dialysis against 5 102 vol of SB appeared to fully retain fluorescence (Fig. 4, trace 3). Again, the addition of GSNA led to a reduction in the fluorescence emission to basal values (Fig. 4, trace 4), while the further addition of GSHNE led to the recovery of an appreciable level of the cofactor in the reduced state (Fig. 4, trace 5).
Fig. 4. The cofactor is retained by hCBR1 during catalysis. NADP-free hCBR1 was loaded with NADPþ as described in Fig. 3 and the corresponding fluorescence spectrum recorded at an enzyme concentration of 2.5 mM (trace 1). Trace 2 refers to the sample after the addition of 25 mM GSHNE. Trace 3 refers to the spectrum of the sample after dialysis against 5 102 vol of SB. This sample showed approximately 80% of the activity measured in sample 2. Trace 4 refers to the sample of trace 3 after the addition of 2.5 mM GSNA. Trace 5 refers to the sample of trace 4 after the addition of 6 mM GSHNE.
Dissecting the two arms of the redox cycle revealed the efficient catalytic response to the substrate addition of the enzyme-bound cofactor in both oxidized and reduced states (Fig. 3 and 4). These results, together with the synchronous generation of the disproportionation products at nominally very low levels of the cofactor, which are conceivably too low for fruitful in and out traffic at the active site (Fig. 1), supported the enzyme’s capacity to firmly retain the cofactor. This applies not only to a structural resting status, but also when the substrate is undergoing transformation. The features of hCBR1 reported in the present study led us to consider the cofactor NADP(H) as a prosthetic group of the enzyme, acting in situ as a donor and acceptor of the hydride ion in order to achieve GSHNE disproportionation. The disproportionation rate of the above-mentioned reaction may possibly be limited by the traffic of substrates at the active site, which is linked both to the intrinsic kinetic parameters and relative concentrations of the substrates themselves. The redox status of the cofactor, on the other hand, likely depends exclusively on the in-situ transformation. Thus, in the case of GSHNE, taking into account the kinetic parameters measured for GSNA for the reduction arm of the disproportionation, the reduction and oxidation reactions should occur at comparable rates near neutrality (pH 7). Thus, even though the KM of the hemiacetal substrate undergoing oxidation is approximately 3-fold higher than that measured for the open aldehyde undergoing reduction [12,14], the limiting step of the GSHNE disproportionation is very likely associated with the relative low concentration of the free aldehyde form. At equilibrium, this accounts for approximately 5% of the total substrate. In terms of the normal flux of the cofactor from the buffer solution to the active site and vice versa, starting for instance from NADPþ, the apparent disproportionation only becomes evident after reaching the appropriate level of the cofactor in the status competent to sustain the opposite reaction (i.e. NADPH). A steady state condition should then be reached in which the two arms of the cycle proceed at the same rate. The same condition may also occur more generally when the functional groups undergoing redox transformation belong to different molecules, as shown here for GSHNE and GSNA.
Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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The release of the cofactor in several different pyridinedependent dehydrogenases is generally reported as the ratelimiting step of the catalytic action [27e30]. Thus, any slowing down of the cofactor traffic at the active site hinders the progression of the on-going reaction. In fact, below a certain level, or when brought to the extreme condition in which the enzyme is unable to release the cofactor, catalysis will not take place. However, when the cofactor retention does not limit the redox recycle ability of the cofactor, as observed in the present study, the reaction may in any case proceed in both directions of the redox process. This is because it is exclusively dependent on the turnover of the two substrates at the active site. Even considering a possible mutual competitive inhibition between the two substrates, this feature could be a significant gain in the catalytic performance of enzymes performing detoxification. In the case of CBR1, for instance, the possible in vivo simultaneous occurrence of the oxidative branch of the disproportionation reaction of GSHNE and of the reduction of other toxic glutathionylaldehyde adducts (such as GSNA) would potentiate the red/ox detoxification ability of the enzyme, i.e. the oxidation of GSHNE hemiacetal to the GSHNA-lactone and the reduction of GSNA to the corresponding alcohol. The oxidation of GSHNE to GSHNA-lactone is thus sustained by the reduction of GSNA, theoretically escaping the effects of both the level and the redox status of the pyridine cofactor present in the solution. In this specific context the detoxification action of CBR1 is further enhanced, as the proinflammatory effect consequent to the generation of one of its products (i.e. GSDHN) is minimized by the more abundant aldehydic competitor substrates (i.e. GSNA). Finally, if we extend the features highlighted for CBR1 to other dehydrogenases involved in cell detoxification, the intra-site redox turnover of the pyridine cofactor may be regarded as a further catalytic device of the enzyme. In fact, the in situ recycling of the cofactor will act as a flywheel of the entry in the catalytic cycle of the free cofactor present in solution. This is because it becomes part of the overall kinetic mechanism (in which some in and out exchanges of the cofactor are possible). This will certainly enhance the detoxifying power of the system. Thus, the cofactor exchange between the enzyme and the solution may be seen as a regulatory step to address the overall direction of the process, rather than a way of replenishing the enzyme with the cofactor. Funding sources
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.090.
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Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group Vito Barracco a, d, 1, Roberta Moschini a, c, 1, Giovanni Renzone b, Mario Cappiello a, c, Francesco Balestri a, c, Andrea Scaloni b, Umberto Mura a, Antonella Del-Corso a, c, * a
University of Pisa, Department of Biology, Biochemistry Unit, Via S. Zeno, 51, Pisa, Italy Proteomics & Mass Spectrometry Laboratory, ISPAAM-CNR, Via Argine, 1085, Napoli, Italy Interdepartmental Research Center Nutrafood ‘‘Nutraceuticals and Food for Health’‘, University of Pisa, Pisa, Italy d PhD Student at the Tuscany Region “Pegaso” PhD School in Biochemistry and Molecular Biology, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2019 Accepted 14 November 2019 Available online xxx
Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed. © 2019 Elsevier Inc. All rights reserved.
Keywords: Carbonyl reductase 1 3-Glutathionyl-4-hydroxynonanal disproportionation NADP(H) prosthetic group
1. Introduction Carbonyl reductase 1 (CBR1), a cytosolic monomeric NADPþdependent secondary alcohol dehydrogenase [E.C.1.1.1.184] is common in mammals [1] and found in several human tissues [2]. The reductase activity of the enzyme is exerted in a variety of substrates, such as eicosanoids, steroids, lipid peroxidation-derived carbonyl compounds, as well as orto- or para-xenobiotic quinone derivatives [3e5]. The active site of CBR1 is quite complex. CBR1 contains a very flexible wide interactive region, which tightly closes around the substrate after binding [6]. A glutathione-binding site, close by the active site, has also been identified [7,8]. This is in
Abbreviations: CBR1, carbonyl reductase 1; DTT, D,L-dithiothreitol; GSDHN, 3glutathionyl-1,4-dihydroxynonane; GSH, reduced glutathione; GSHNA-lactone, 3glutathionyl-4-hydroxynonanoic-g-lactone; GSHNE, 3glutathionyl-4hydroxynonal; GSNA, 3-glutathionyl-nonanal; hCBR1, human recombinant CBR1; HNE, 4-hydroxy-2-nonenal. * Corresponding author. Department of Biology, Biochemistry Unit, via S. Zeno 51, 56127, Pisa, Italy. E-mail address: [email protected] (A. Del-Corso). 1 These authors contributed equally to the work.
addition to the expected interplay of functional residues at the active site of CBR1, such as Cys227 which in its negative thiolate status, orients the substrate [9,10] and Trp229, Ala235 and Met241 involved in the binding of the hydrophobic moieties of substrates [5]. The ability of CBR1 to intervene as a reductase not only on keto groups, as expected for a secondary alcohol dehydrogenase, but also on aldehydic groups, is still an open issue. Although aldehydes (including 4-hydroxy-2-nonenal - HNE) are poor substrates for the enzyme [5], a 10% reduction of the aldehydic group, together with an oxo-reduction, has been reported when 4-oxo-trans-2-nonenal was used as a substrate [11]. CBR1 appeared to be much more efficient in catalyzing the reduction of glutationylated aldehydes [12], and this evidence is conceivably supported by the abovementioned GSH binding site. The oxo group in position 9 of prostaglandin-A1 used as substrate for the enzyme, was reduced only on the glutathionylated adduct. The latter is also able to inhibit the 15-hydroxyprostaglandin dehydrogenase activity of CBR1 on prostaglandin-B1 [7,10]. The strong effect of the glutathionyl moiety of substrates in addressing the substrate on the active site makes the effect of
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Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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Cys227 any more critical. However, this did not apply to S-nitrosoglutathione, whose efficient reduction by the enzyme is subjected to Cys227 control [7,13]. All this evidence underlines the complexity of the substrate allocation at the active site of CBR1. The ability of CBR1 to intervene on 3-glutathionyl-4hydroxynonanal (GSHNE) metabolism both as a dehydrogenase, on the hemiacetal hydroxyl group [14], and as a reductase, on the free aldehydic carbonyl group [12], catalyses an overall disproportionation reaction of the molecule at sub-stoichiometric levels of the pyridine cofactor [15]. Thus, sustained by a NADPþ/NADPH recycle, GSHNE was both oxidized to the corresponding 3glutathionyl-4-hydroxynonanoic-g-lactone (GSHNA-lactone), ready for cell extrusion [16] and reduced to 3-glutathionyl-1,4dihydroxynonane (GSDHN), a pro-inflammatory molecular signal [17]. This paper highlights that CBR1 may act as catalyst of the disproportionation reaction without any cofactor exchange with the solution, being able of a redox recycle of a stoichiometric amount of bound cofactor.
2.4. Liquid chromatography-mass spectrometry analysis of GSHNE reaction products Assay mixtures containing GSHNE (prepared as described in Section 2.5) were incubated in different conditions either in the absence or in the presence hCBR1, and analysed in order to determine the reaction extent on a time-course basis. Identical aliquots sampled at different times were immediately frozen, lyophilized and then stored at 80 C, until further use. Samples were dissolved in 0.1% trifluoroacetic acid and subjected to nanoLC-ESI-LIT-MS/MS analysis with an LTQ XL mass spectrometer (ThermoFisher, USA) equipped with a Proxeon nanospray source, which was connected to an UltiMate 3000 RSLC nano-liquid chromatographer (ThermoFisher, USA) [14,15]. Analyte mixtures were resolved, and the corresponding spectra were acquired [14,15]. Assignment of MS/MS spectra to specific glutathione derivatives and semi-quantitative measurement of the reaction products were then carried out [14,15]. 2.5. Preparation of glutathionylated substrates
2. Materials and methods 2.1. Materials All reagents, solvents and filtration devices used were as previously described [14].
GSHNE and 3-glutathionylnonanal (GSNA) were prepared by incubating 1.5 mM GSH in 50 mM sodium phosphate buffer, pH 7.4, with 1 mM of either HNE or trans-2-nonenal, respectively [14]. Taking into account the disappearance of HNE and trans-2-nonenal (by measurements at 224 nm), the residual GSH [18] and the spontaneous oxidation of GSH [19], a stoichiometric consumption of GSH and HNE was observed, with less than 10% residual alkenals. Both thiolated adducts were stored at 80 C until used.
2.2. Enzyme assay, expression and purification
2.6. Other methods
The activity of human recombinant carbonyl reductase 1 (hCBR1) was determined at 37 C in an assay mixture containing, in 50 mM sodium phosphate buffer pH 8.4, 0.18 mM NADPþ and 0.1 mM GSHNE as substrate [12]. The reaction was monitored following the increase in absorbance at 340 nm linked to the reduction of NADPþ. One Unit of enzyme activity is the amount of enzyme that catalyses the conversion of 1 mmol of substrate/min in the specified assay conditions. hCBR1 was expressed and purified to electrophoretic homogeneity as previously described [12]. Purified hCBR1 samples, at a concentration of 0.15 mg/mL (5 mM, calculated on the basis of a molecular mass of 31 KDa) and displaying a specific activity of 50 U/ mg, were stored at 80 C in a 10 mM sodium phosphate buffer pH 7.0 (Standard Buffer, SB) containing 1 mM D,L-dithiothreitol (DTT), 0.05 mM NADPþ, 0.75 M NaCl and 33% w/v glycerol. Before use, the enzyme was extensively dialyzed against SB.
Protein concentration was determined according to the Bradford method [20]. Fluorescence spectra were obtained with an FP-6500 spectrofluorometer (Jasco, Easton, MD, USA) using an excitation wavelength of 345 nm. Emission fluorescence was measured at wavelengths of between 400 and 600 nm.
2.3. Dialysis protocols In order to obtain a NADP-free hCBR1, 3 mM of purified hCBR1 was dialyzed at 4 C through ultra-membrane filters (10 kDa cutoff) against 2 103 vol of SB supplemented with 1.5 M NaCl. A second dialysis step against 4 103 vol of SB in the above conditions was then carried out. The enzyme concentration appeared critical for the recovery of the enzyme activity after dialysis. In order to ensure a recovery higher than 80%, both in terms of protein concentration and specific activity, the protein concentration was never allowed to be lower than 0.6 mM throughout the dialysis. NADP-free hCBR1 (3 mM) was loaded with NADPþ or NADPH by incubation for 15 min at 25 C in the presence of 30 mM of either NADPþ or NADPH, respectively, followed by dialysis against 4 103 vol of SB.
3. Results and discussion When a highly-purified preparation of hCBR1 (63 nM) extensively dialyzed against 4 103 vol of SB (leading to a residual nominal cofactor level in the solution of 0.16 nM, i.e. less than 0.25% with respect to the enzyme concentration) was added to a GSHNE solution, both the oxidation and reduction of the adduct occurred, although the cofactor was nominally absent in the assay. As shown in Fig. 1A, the disappearance of the substrate and the parallel generation of both GSHNA-lactone and GSDHN occurred at a detectable rate, halving the substrate concentration within 20 min. No significant differences were observed in the reaction rate when the above-described solution was supplemented by NADPþ up to a nominal concentration of the cofactor of 63 nM, accounting for a cofactor/enzyme molar ratio of 1 (data not shown). This could be due to either a disproportionation reaction of GSHNE occurring in the absence of the cofactor, or alternatively, to the fact that, despite the very extensive dialysis step, the cofactor was firmly retained by the enzyme and recycled on it while performing catalysis. To the best of our knowledge, no structural evidence is available to support the ability of the enzyme to bind two substrate molecules at the active site leading to their disproportionation reaction. The involvement of the cofactor trapped on the active site, while catalysis is taking place, thus appeared to be the most likely explanation. Indeed, evidence of NADP bound on hCBR1 following purification has already been reported [21]. The ability of pyridine cofactors to tightly bind enzymes is not infrequent [22,23], and the
Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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Fig. 1. Time-course of GSHNE transformation catalyzed by hCBR1. GSHNE (100 mM) was incubated at 37 C in a sodium phosphate buffer 50 mM pH 6.2 in various conditions in the presence of 94 mU/mL (63 nM) of purified hCBR1 and the reaction mixture analysed by mass spectrometry (see Section 2.4). Plots are reported of the peak areas of the molecular species at m/z values 464.25 (GSHNE - circles), 466.25 (GSDHN - squares) and 462.23 (GSHNA-lactone - triangles), as obtained by integrating the extracted ion currents relative to nanoLC-ESI-MS runs at different incubation times. The experimental points are reported with the corresponding error bars, and represent the average of at least three independent measurements. Panel A: the incubation was performed with the enzyme previously dialyzed against 4 103 vol of SB. Panel B: the incubations were performed with the NADP-free hCBR1 (see Section 2.3). Curves marked with 1, 2 and 3 refer to incubations performed supplementing the solution with zero, 63 nM and 313 nM NADPþ, respectively.
implications of such an event on the functional features of the involved enzymes have been underlined [24]. Such evidence for CBR1 is the rational base to further the apparent disproportionation reaction of GSHNE. Gaining key evidence to support this point entailed assessing presence of the cofactor on hCBR1 for catalysis. The possibility of removing the bound cofactor, whenever present, from the enzyme was further evaluated by adopting more severe dialysis conditions. Thus, the application of the dialysis protocol (see Section 2.3) to hCBR1 led to the complete abolishment of the disproportionation reaction of the substrate (Fig. 1B). The disappearance of the substrate and the parallel formation of GSDHN and GSHNA-lactone occurred only following the addition of low concentrations of the cofactor, accounting for a cofactor/enzyme molar ratio from 1 to 5. Since the GSHNE transformation far exceeded the concentration of the pyridine cofactor, these results confirmed the occurrence of an overall disproportionation reaction, ruling out the potential of any NADP(H)-independent ability of hCBR1 to catalyze this reaction. The efficiency of the bound cofactor in intervening in the redox step was then assessed. This was achieved by exploiting the emission fluorescence spectrum shown by NADPH, but not by NADPþ, with a maximum emission at 460 nm when excited at 345 nm. Following the interaction of the cofactor with the protein [25,26], the NADPH spectrum showed a modest hypsochromic shift and an increase in emission intensity. Fig. 2 shows the differential fluorescence spectrum between the trace recorded in the case of a hCBR1 solution (1.2 mM) saturated with NADPH, and the counterpart of an equally concentrated NADPH solution in the buffer. The inset in Fig. 2 reports the saturation process of hCBR1 by the cofactor as the differential emission intensity at 460 nm, which resulted from the gradual addition of NADPH to the enzyme. No fluorescence emission was measured after the enzyme was saturated with NADPþ (Fig. 2, trace 4). This confirms the effectiveness of this methodological approach to
3
Fig. 2. Loading of hCBR1 with NADPH. NADP-free hCBR1 (see Section 2.4) (1.2 mM) in SB was supplemented with increasing concentrations of NADPH. The corresponding fluorescence emission spectra (345 nm excitation wavelength) were recorded. Traces 1 and 2 represent the spectrum of 32 mM NADPH in SB alone or in the presence of 1.2 mM hCBR1, respectively. The dotted line refers to the differential spectrum obtained by subtracting trace 1 from trace 2. Traces 3 and 4 refer to the spectra of the NADP-free hCBR1 before and after the addition of 32 mM NADPþ, respectively. The inset shows the differential emission fluorescence at 450 nm as a function of the cofactor/enzyme ratio obtained upon the addition of exogenous NADPH.
follow the redox status of the cofactor. To verify the involvement of the bound cofactor in the disproportionation reaction, the enzyme deprived of the endogenous cofactor (i.e. NADP-free hCBR1, as defined in 2.3) was loaded with either NADPþ or NADPH. Following an extensive dialysis, it was then subjected to a spectrofluorometric analysis in various conditions thereby allowing the enzyme to exert catalysis. The enzyme loaded with NADPH (as reported in Fig. 2) was thus subjected to extensive dialysis against 4 103 vol of SB, and the corresponding fluorescence spectrum was measured. This spectrum (Fig. 3A, trace 1) coincided with the calculated differential spectrum in Fig. 2, and thus indicated a complete retention of the loaded cofactor after dialysis. The addition of 1.6 mM GSNA to the solution (corresponding to a two-fold concentration with respect to the enzyme) (Fig. 3A, trace 2) led to a very rapid (a few seconds) decrease in fluorescence, possibly associated with the NADPH-dependent reduction of the substrate. Likewise, the in situ reduction of the bound NADPþ was followed by adding GSHNE to hCBR1 pre-loaded with NADPþ (Fig. 3B, traces 1 to 5). A gradual increase in the intensity of the fluorescence was observed a few seconds after each addition of GSHNE, which reached a maximum at a substrate level that was approximately 8fold the enzyme concentration (trace 5). This fluorescence emission represented 65% of the NADPH load capacity of the enzyme (Fig. 3A, trace 1). It was compatible with the in situ reduction of the bound NADPþ by a substrate such as GSHNE, which may undergo both oxidation and reduction reactions. When the solution was supplemented with GSNA at a substrate/ enzyme molar ratio of 2, a very rapid decrease in fluorescence was observed (Fig. 3B, trace 6), which was compatible with NADPH oxidation. Note that over time the intensity of the fluorescence slowly started to recover (Fig. 3B, inset), likely due to presence of residual GSHNE in the mixture. Implementing the GSHNE concentration and thus favouring the oxidative arm of the disproportionation cycle, led to a rapid increase in the emission intensity up to approximately 50% of the initial value (Fig. 3B, trace 5). In order to rule out the possibility of a mobilization of the bound
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Fig. 3. Separation of the two arms of the GSHNE disproportionation reaction. Panel A: hCBR1 saturated with NADPH, as described in Fig. 2, was extensively dialysed against 4 103 vol of SB, and the corresponding spectrum was recorded at an enzyme concentration of 0.8 mM (trace 1). Trace 2 refers to the enzyme sample supplemented with 1.6 mM GSNA. Panel B: NADP-free hCBR1 was saturated with NADPþ (see Section 2.3), and the corresponding spectrum was recorded at an enzyme concentration of 0.8 mM (trace 1). Traces 2, 3, 4, 5 refer to the emission spectra recorded after the gradual addition of GSHNE to the enzyme solution to obtain final concentrations of 0.8 mM, 1.6 mM, 3.2 mM and 6.4 mM, respectively. Trace 6 refers to the further addition of 1.6 mM GSNA to the sample of trace 5. The inset shows the slow recovery of fluorescence emission from the sample of trace 6, which was enhanced by again supplementing the solution with 6.4 mM GSHNE at the time indicated by the arrow.
cofactor during catalysis, a dialysis step was inserted between the forward and reverse reactions. This step was designed to remove the excess GSHNE and, if present, the mobilized cofactor. After the in situ reduction of NADPþ by GSHNE (Fig. 4, trace 2), the recovered hCBR1 activity (85% of the initial value) after dialysis against 5 102 vol of SB appeared to fully retain fluorescence (Fig. 4, trace 3). Again, the addition of GSNA led to a reduction in the fluorescence emission to basal values (Fig. 4, trace 4), while the further addition of GSHNE led to the recovery of an appreciable level of the cofactor in the reduced state (Fig. 4, trace 5).
Fig. 4. The cofactor is retained by hCBR1 during catalysis. NADP-free hCBR1 was loaded with NADPþ as described in Fig. 3 and the corresponding fluorescence spectrum recorded at an enzyme concentration of 2.5 mM (trace 1). Trace 2 refers to the sample after the addition of 25 mM GSHNE. Trace 3 refers to the spectrum of the sample after dialysis against 5 102 vol of SB. This sample showed approximately 80% of the activity measured in sample 2. Trace 4 refers to the sample of trace 3 after the addition of 2.5 mM GSNA. Trace 5 refers to the sample of trace 4 after the addition of 6 mM GSHNE.
Dissecting the two arms of the redox cycle revealed the efficient catalytic response to the substrate addition of the enzyme-bound cofactor in both oxidized and reduced states (Fig. 3 and 4). These results, together with the synchronous generation of the disproportionation products at nominally very low levels of the cofactor, which are conceivably too low for fruitful in and out traffic at the active site (Fig. 1), supported the enzyme’s capacity to firmly retain the cofactor. This applies not only to a structural resting status, but also when the substrate is undergoing transformation. The features of hCBR1 reported in the present study led us to consider the cofactor NADP(H) as a prosthetic group of the enzyme, acting in situ as a donor and acceptor of the hydride ion in order to achieve GSHNE disproportionation. The disproportionation rate of the above-mentioned reaction may possibly be limited by the traffic of substrates at the active site, which is linked both to the intrinsic kinetic parameters and relative concentrations of the substrates themselves. The redox status of the cofactor, on the other hand, likely depends exclusively on the in-situ transformation. Thus, in the case of GSHNE, taking into account the kinetic parameters measured for GSNA for the reduction arm of the disproportionation, the reduction and oxidation reactions should occur at comparable rates near neutrality (pH 7). Thus, even though the KM of the hemiacetal substrate undergoing oxidation is approximately 3-fold higher than that measured for the open aldehyde undergoing reduction [12,14], the limiting step of the GSHNE disproportionation is very likely associated with the relative low concentration of the free aldehyde form. At equilibrium, this accounts for approximately 5% of the total substrate. In terms of the normal flux of the cofactor from the buffer solution to the active site and vice versa, starting for instance from NADPþ, the apparent disproportionation only becomes evident after reaching the appropriate level of the cofactor in the status competent to sustain the opposite reaction (i.e. NADPH). A steady state condition should then be reached in which the two arms of the cycle proceed at the same rate. The same condition may also occur more generally when the functional groups undergoing redox transformation belong to different molecules, as shown here for GSHNE and GSNA.
Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090
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The release of the cofactor in several different pyridinedependent dehydrogenases is generally reported as the ratelimiting step of the catalytic action [27e30]. Thus, any slowing down of the cofactor traffic at the active site hinders the progression of the on-going reaction. In fact, below a certain level, or when brought to the extreme condition in which the enzyme is unable to release the cofactor, catalysis will not take place. However, when the cofactor retention does not limit the redox recycle ability of the cofactor, as observed in the present study, the reaction may in any case proceed in both directions of the redox process. This is because it is exclusively dependent on the turnover of the two substrates at the active site. Even considering a possible mutual competitive inhibition between the two substrates, this feature could be a significant gain in the catalytic performance of enzymes performing detoxification. In the case of CBR1, for instance, the possible in vivo simultaneous occurrence of the oxidative branch of the disproportionation reaction of GSHNE and of the reduction of other toxic glutathionylaldehyde adducts (such as GSNA) would potentiate the red/ox detoxification ability of the enzyme, i.e. the oxidation of GSHNE hemiacetal to the GSHNA-lactone and the reduction of GSNA to the corresponding alcohol. The oxidation of GSHNE to GSHNA-lactone is thus sustained by the reduction of GSNA, theoretically escaping the effects of both the level and the redox status of the pyridine cofactor present in the solution. In this specific context the detoxification action of CBR1 is further enhanced, as the proinflammatory effect consequent to the generation of one of its products (i.e. GSDHN) is minimized by the more abundant aldehydic competitor substrates (i.e. GSNA). Finally, if we extend the features highlighted for CBR1 to other dehydrogenases involved in cell detoxification, the intra-site redox turnover of the pyridine cofactor may be regarded as a further catalytic device of the enzyme. In fact, the in situ recycling of the cofactor will act as a flywheel of the entry in the catalytic cycle of the free cofactor present in solution. This is because it becomes part of the overall kinetic mechanism (in which some in and out exchanges of the cofactor are possible). This will certainly enhance the detoxifying power of the system. Thus, the cofactor exchange between the enzyme and the solution may be seen as a regulatory step to address the overall direction of the process, rather than a way of replenishing the enzyme with the cofactor. Funding sources
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This work was supported by Pisa University, PRA 2017. [24]
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.090.
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Please cite this article as: V. Barracco et al., Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.090