Inactivation of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Chemico-Biological Interactions 191 (2011) 159–164

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Inactivation of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide Jesús A. Rosas-Rodríguez, Elisa M. Valenzuela-Soto ∗ Coordinación de Ciencia de los Alimentos, Centro de Investigación en Alimentación y Desarrollo A.C., P.O. Box 1735, Hermosillo, Sonora 83000, Mexico

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Article history: Available online 4 February 2011 Keywords: Cellular stress response Kidney Betaine aldehyde dehydrogenase Inactivation Hydrogen peroxide

a b s t r a c t Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H2 O2 (0–500 ␮M) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD+ (10–50 ␮M) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 ␮M) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD+ . Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD+ binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289). © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction One of the most important functions of renal medulla cells is the concentration of urine. An efficient urinary concentration mechanism requires increased NaCl, increased urea concentrations and hypoxia [1–3]; the latter is required to achieve adequate urinary concentration [1,4]. It has been demonstrated that medullary cells with high NaCl concentrations induced an increase in the concentration of reactive oxygen species (ROS) in mouse thick ascending limb (mTAL) of Henle and in cultivated cells (HEK293) [5–7]. ROS are present at low concentrations and are distributed among different compartments of the cell; however, oxidative stress results when the balance between reactive species and antioxidants is lost. It is known that the most abundant ROS in renal cells are the superoxide radical (O2 − ) and hydrogen peroxide (H2 O2 ). Hydrogen peroxide is an oxygen metabolite produced during the complete

Abbreviations: BA, betaine aldehyde; BADH, betaine aldehyde dehydrogenase; DTT, dithiothreitol; GB, glycine betaine; GSH, reduced glutathione; pkBADH, porcine kidney betaine aldehyde dehydrogenase; ROS, reactive oxygen species. ∗ Corresponding author. Tel.: +52 662 289 2400; fax: +52 662 280 0381. E-mail address: [email protected] (E.M. Valenzuela-Soto). 0009-2797/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.01.030

reduction of oxygen to H2 O, and it is biologically important due its ability to generate the highly reactive hydroxyl radical [8]. ROS react principally with amino acids via hydrogen abstraction (aliphatic amino acids), electron transfer (sulfur-containing amino acids) and addition (aromatic amino acids). ROS react with cysteine to form sulfenic acid and cystine [9]. Betaine aldehyde dehydrogenase (betaine aldehyde: NAD+ oxidoreductase; EC 1.2.1.8; and BADH) from mammals is an enzyme that belongs to the superfamily of aldehyde dehydrogenases (ALDH9) [10,11]. BADH catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB). GB acts as an osmolyte in animals, plants and bacteria [12–14]; in mammals it is synthesized and accumulated in renal cells, and protects them from hyperosmotic stress [14,15]. GB also functions as the methyl donor in methionine synthesis [16,17]. GB and homocysteine are also substrates of betaine-homocysteine methyl transferase (BHMT: EC 2.1.1.15) [18,19]. Adequate BADH function provides enough GB to metabolize 25% of homocysteine, preventing development of hyperhomocysteinemia. Homocysteine accumulation is associated with cardiovascular disease, thus, GB plays an important role in decreasing the risk of heart disease [20]. All BADHs studied to date (mostly from Pseudomonas aeruginosa) include a highly conserved cysteine in their active site

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(catalytic cysteine) and other amino acids important for accurate protein folding [10,21–23]. The sulfur-containing residues cysteine and methionine are the most sensitive of the 20 amino acids potentially targeted by ROS [24,25]. The compositional characteristics of BADH make it a potential target for modification by H2 O2 modifications. In this research, the impact of H2 O2 on porcine kidney betaine aldehyde dehydrogenase (pkBADH) stability was analyzed. The effect of ligands on the kinetics of enzyme inactivation by hydrogen peroxide, and the reversibility of this inactivation was also evaluated. 2. Material and methods 2.1. Chemicals Betaine aldehyde chloride (BA), glycine betaine (GB), NAD+ , NADH, EDTA, HEPES buffer and hydrogen peroxide were purchased from Sigma. N-6-hexyl-AMP-agarose was purchased from Jena Bioscience. All other analytical grade chemicals were from standard suppliers. 2.2. Enzyme purification and activity assay BADH was purified from porcine kidneys as previously reported [26] except that N-6-hexyl-AMP-agarose was used as the affinity matrix. Enzyme activity was assayed by following the reduction of NAD+ at 340 nm using a spectrophotometer (Ultrospec 4000) at 25 ◦ C. The standard assay system contained 100 mM Hepes-KOH buffer, pH 8.0; 0.1 mM EDTA, 0.5 mM BA, and 1.0 mM NAD+ in a total volume of 0.4 ml. The reaction was started by adding the enzyme. Each determination was performed in duplicate. 2.3. Inactivation assays Concentrations of stock H2 O2 were determined by measuring the absorbance at 240 nm (ε240 = 44 M−1 cm−1 ). BADH (10 ␮M) was incubated with varying H2 O2 concentrations (25–500 ␮M) at 25 ◦ C. Aliquots were withdrawn at different time intervals, and the residual activity was assayed immediately as described above. The effect of ligands was evaluated by incubating the enzyme with 100 ␮M H2 O2 plus NAD+ (0.01–50 ␮M), BA (0.05 and 0.5 mM), GB (100 mM) or NADH (0.01–0.5 mM) under the same conditions as used in the assays without substrates. The ligands were added prior to the addition of H2 O2 . Rate constants for inactivation were calculated by fitting the data to a single exponential decay equation using OriginPro 8.0 (OriginLab, Northampton, MA, USA). 2.4. Reactivation assays BADH was inactivated by H2 O2 (2 mM) after 150 min incubation. The mixture was then incubated at 25 ◦ C with 10 mM DTT, 10 mM 2mercaptoethanol or 10 mM GSH. Aliquots were drawn at different time intervals, and the residual activity was assayed immediately as described in the activity assay. Reactivation data were fitted to a single exponential growth equation using OriginPro 8.0. 2.5. HPLC mass spectrometry Enzyme incubated with 100 ␮M H2 O2 and enzyme incubated with 50 ␮M NAD+ and 100 ␮M H2 O2 were sent to the Quebec Genomics Center for mass spectrometry (LC/MS/MS) analysis. The BADH protein sample was desalted, purified and buffer exchanged using a centrifugal filter device (Microcon). Thereafter, pkBADH was digested with trypsin. Prior to the digestion, reduction or alkylation of cysteines was not carried out so that the hydrogen

peroxide oxidation products could be analyzed. A protein bank containing only BADH was built. For the mass spectrometry analysis, an inclusion list was created containing the BADH active site peptide (GALMANFLTQGEVCCNGTR) with all combinations of oxidized and unoxidized M and C residues. Peptides were preferentially analyzed according to the inclusion list. For analysis of the results, the oxidation (+16) of the Cys, Met, His and Trp residues were used as the variable modification. The resulting data were analyzed using ScaffoldTM 2007 (Proteome Software Inc.) and were visualized through structural models using PyMOL Molecular Graphics System (DeLano Scientific LLC). 2.6. Molecular modeling To obtain the pkBADH structure model, we used the MOE (Molecular Operating Environment) v2007.09 software from Chemical Computing Group. The pkBADH structure was elaborated using other BADH models deposited in the RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do). Two of the six BADH models that are in the database, cod liver (1A4S, doi:10.2210/pdb1a4s/pdb) and Escherchia coli (1WNB, doi:10.2210/pdb1wnb/pdb), showed high levels of identity (69% and 38%, respectively) with pkBADH, so these two BADH sequences were used to construct the pkBADH model. The model obtained was analyzed with two types of molecular visualization software: PyMOL 1.1 (by DeLano Scientific LLC) and WinCoot 0.3.3 (by Paul Emsley & Kevin Cowtan). 3. Results 3.1. Inactivation studies Hydrogen peroxide at low concentration (100 ␮M) poorly inactivated pkBADH; however as shown in Fig. 1 a 25% inactivation occurred at 500 ␮M H2 O2 . The enzyme was incubated with 2 mM H2 O2 during 150 min to reach 85% inactivation (data not shown). Enzyme inactivation was monophasic and occurred in a timeand dose-dependent manner (Fig. 1). The pseudo-first order rate constants of inactivation (kobs ) were dependent on the H2 O2 concentration (Fig. 1B), but the kinetics are complex and not described by a simple bimolecular reaction. 3.2. Effect of ligands on pkBADH inactivation by H2 O2 pkBADH was incubated with a fixed concentration of H2 O2 (100 ␮M) and with varying concentrations of NAD+ (10–50 ␮M). In the absence of NAD+ , pkBADH was inactivated 17% by H2 O2 , whereas in the presence of NAD+ , inactivation was 60% (Fig. 2A). The pseudo-first order rate constants of inactivation (kobs ) were dependent on the NAD+ concentration, but again not as for a simple bimolecular reaction (Fig. 2B). Preincubation of the enzyme with NAD+ (10 ␮M) for 1, 5, 10 or 60 min before adding 100 ␮M H2 O did not affect the inactivation by hydrogen peroxide (data not shown). The substrate betaine aldehyde and the products glycine betaine and NADH were used in the inactivation assay to determine whether they have any effect on enzyme inactivation. The presence of BA (Fig. 2C) or GB (Fig. 2D) did not increase pkBADH inactivation by H2 O2 (20% inactivation with and without ligands), and no protective effect was detected under inactivation conditions. NADH had no effect on pkBADH inactivation by H2 O2 (data not shown). 3.3. pkBADH molecular modeling To understand and visualize the possible targets of hydrogen peroxide in pkBADH, we first needed to know how the protein domains interact at the molecular level and which residues belong

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Fig. 1. Time courses of the inactivation of pkBADH by hydrogen peroxide. (A) Enzyme incubated with 0–500 ␮M H2 O2 . The lines are the best fit of the inactivation data to a single exponential decay equation. (B) Dependence of the pseudo-first order rate constant for the inactivation on the H2 O2 concentration.

Fig. 2. Effect of enzyme ligands on the inactivation of pkBADH by hydrogen peroxide. pkBADH was incubated with 100 ␮M H2 O2 in the presence of the following ligands: (A) 0 to 50 ␮M NAD+ ; (C) 50 and 500 ␮M BA; and (D) 100 mM GB. The lines are the best fit of the inactivation data to a single exponential decay equation. (B) Dependence of the rate constants for inactivation on the NAD+ concentration.

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Fig. 3. Model of a pkBADH subunit showing the functional domains and the active site. Enzyme residues involved in substrate binding are shown in the magnified area (labeled). Vicinal cysteines in the catalytic site are labeled.

to each functional domain. To identify these sites, we used a resource called iPfam [27], which is hosted by the Pfam UK website, to analyze the pkBADH sequence. The analysis revealed one significant iPfam interaction, predicting that amino acids E254 and C288 were part of the active site. This result was interesting because both of these amino acids were involved in substrate binding, E254 to NAD+ and C288 to betaine aldehyde. Using PyMOL software, we visualized the NAD+ and BA binding domain of pkBADH. It was possible to identify the amino acids that make up the catalytic cavity (Fig. 3). 3.4. HPLC mass spectrometry Mass spectrometry analysis of inactivated pkBADH showed that active site cysteines were not oxidized by incorporation of oxygen, whereas several other residues incorporated one oxygen per residue. Notably, the amino acids oxidized by H2 O2 in the presence and absence of the coenzyme differed. Residues M278, M243, M241 and H335 of the enzyme incubated with H2 O2 were oxidized. When the enzyme was incubated with NAD+ and H2 O2 , residues M94, M327 and M278 were oxidized. pkBADH molecular modeling allowed us to identify the position of the residues oxidized by H2 O2 . Residues M278, M243, M241 and H335 are near the coenzyme binding site and residues M94, M327 and M278 are located proximal to the BA binding site (Fig. 4).

active site (pkBADH C288), but pkBADH has another vicinal cysteine (C289), suggesting that C288 and C289 could react with H2 O2 to form cystine, thus affecting the binding of the betaine aldehyde substrate. The results show that pkBADH was inactivated by about 17% when it was exposed to 100 ␮M hydrogen peroxide (Fig. 1). However, when pkBADH was exposed to 100 ␮M hydrogen peroxide in the presence of NAD+ , inactivation approached 60% (Fig. 2A). The partial inactivation can result from complete modification of one or more residues producing an enzyme with altered kinetic constants or partial modification of enzyme with complete inactivation, or both. The complex kinetics suggest that the inactivation is not simple, but do not allow to distinguish among the possibilities. Although several methionine residues were oxidized, these residues probably are not directly involved in catalysis, and we think that a more likely explanation is that active site cysteine residues are involved. It appears that the presence of oxidized

3.5. pkBADH reactivation studies pkBADH inactivated by 2 mM H2 O2 was incubated with 10 mM DTT, 10 mM 2-mercaptoethanol or 10 mM GSH. Enzyme incubated with 2-mercaptoethanol and DTT recovered 50% and 85% of its activity, respectively (Fig. 5). GSH was unable to restore enzyme activity (Fig. 5). No significant pkBADH activity changes were obtained after long incubation periods. 4. Discussion H2 O2 has been shown to reversibly inactivate reactive cysteinecontaining enzymes both in vitro and in vivo due to the formation of sulfenic groups or cystine (disulfide bond between vicinal cysteines) [24,28–30]. All BADHs are known to have a cysteine in the

Fig. 4. pkBADH ligand binding site model showing residues oxidized by hydrogen peroxide. Enzyme residues M241, M243 and H335 were oxidized by H2 O2 in the absence of coenzyme. The incubation of pkBADH with NAD+ resulted in the oxidation of residues M94, M276 and M327.

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Fig. 5. Kinetics of the reactivation of pkBADH inactivated by hydrogen peroxide. Enzyme inactivated by 2 mM H2 O2 to a residual activity of 15% over a 150 min reaction was incubated with 10 mM DTT, 10 mM 2-mercaptoethanol or 10 mM GSH. The lines are the best fit of the inactivation data to a single exponential decay equation.

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sequence includes 16 cysteines per monomer distributed in specific areas of its structure. The NAD+ binding domain is formed mainly by polar neutral amino acids, except for E254, which is involved in the proton relay system (E252 in PaBADH), and W156, which interacts with the phosphate group of the NAD+ molecule [21]. The fact that pkBADH inactivation increased in the presence of oxidized coenzyme, was reversed by DTT and did not involve the addition of oxygen to the catalytic cysteine suggesting that the oxidation caused by H2 O2 resulted in disulfide bridge formation between C288 and C299. Enzyme inactivation has physiological relevance due to the several roles played by GB in mammalian cells during oxidative stress [34–36]. In summary, we found evidence that pkBADH is susceptible to H2 O2 -induced inactivation. The presence of coenzyme NAD+ increased the extent of inactivation. Enzyme inactivation in presence of NAD+ was detected at relatively low H2 O2 concentrations. The results point to disulfide bridge formation between C288 and C289. The inactivation observed in this study negatively affects the function of pkBADH and consequently the synthesis of glycine betaine. In addition, knowing the specific modifications of redox proteins caused by ROS contributes to the better understanding of the changes that occur during oxidative stress and gives insight into how oxidative stress can be prevented. Conflicts of interest statement

coenzyme increases the accessibility or reactivity of the catalytic cysteine toward hydrogen peroxide. BADH from Pseudomonas aeruginosa and amaranth leaves showed similar behavior in the presence of NAD+ and thiol-specific reagents [22,25]. The increase in H2 O2 -induced inactivation of pkBADH produced by NAD+ might be relevant in vivo because it is likely that an important proportion of the total enzyme exists as a binary complex with the oxidized coenzyme. In addition, kidney medulla cells are under hyperosmotic and hypoxic conditions that can lead to an increase in the H2 O2 concentration [1–4]. The absence of an effect on hydrogen peroxide-induced inactivation of pkBADH in presence of BA, GB or NADH is in accordance with the pkBADH mechanism, suggesting that the aldehyde substrate cannot bind to the enzyme prior to coenzyme binding and that the catalytic site is not differentially exposed upon BA binding [31]. The lack of oxygen addition to the catalytic cysteine, as demonstrated by mass spectroscopy analysis rules out sulfenic, sulfinic or sulfonic acid formation and suggests that a disulfide bridge is formed between vicinal cysteines (C288–C289). Carugo et al. [32] analyzed the structure of 20 proteins forming disulfide bridges between vicinal cysteines and found that nearly 90% of them showed a type ␤-turn conformation structure. The structure model of pkBADH was compared with that of methanol dehydrogenase (pdb 1g72) to look for type VIII folding, given that this enzyme was one of the proteins analyzed by Carugo et al. Both models were very similar, thus we think that pkBADH exhibits the type VIII ␤-turn, folding that allows for disulfide bridge formation. pkBADH inactivation by H2 O2 was reversed by DTT (85%) but not by GSH (Fig. 5). It is interesting that GSH, the only physiological antioxidant tested in this assay, was unable to restore pkBADH activity. The failure of GSH to reactivate other BADHs inactivated by thiol-specific reagents has been previously reported [25,33]; however, the reason for this failure is not yet clear and may be related to the larger size of GSH, which prevents it from accessing the active site. The three dimensional model of pkBADH (Fig. 3) developed in this study allowed us to identify and locate the potential targets of H2 O2 oxidation. The presence of catalytic cysteine C288 (C286 in P. aeruginosa) and vicinal cysteine C289 in the pkBADH active site makes them potential targets for H2 O2 attack (Fig. 3). The pkBADH

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