Aldo-keto reductase from Helicobacter pylori – role in adaptation to growth at acid pH Denise Cornally 1 , Blanaid Mee 1 , Ciara ´ n MacDonaill 1 , Keith F. Tipton 2 , Dermot Kelleher 3 , Henry J. Windle 3 and Gary T. M. Henehan 1 1 School of Food Science and Environmental Health, Dublin Institute of Technology, Ireland 2 Department of Biochemistry, Trinity College Dublin, Ireland 3 Department of Clinical Medicine and Institute of Molecular Medicine, Trinity College Dublin, Ireland Helicobacter pylori is one of the most common human pathogens, and has been strongly linked with chronic gastritis, ulceration, and gastric adenocarcinoma. It has been estimated that approximately 90–95% of duodenal ulcers in Europe originate from an H. pylori infection [1]. In view of the importance of H. pylori as a human pathogen, it is important to understand the mechanisms whereby it colonizes the gastric mucosa. A key factor required for colonization of the gastric mucosa is the organism’s ability to survive in acidic environments. Survival at acid pH is facilitated by the presence of a urease enzyme. However, a report of the isolation of a pathogenic urease-negative strain of this organism suggests that other mechanisms are also important [2]. Ancillary genes required for growth at acid pH values have been identified using a random insertional mutagenesis technique [3]. Several of these were proteins of unknown function, including an aldo-keto reductase (AKR). The role of the AKR in acid adapta- tion was not clear, as a direct disruption of the H. pylori AKR gene was not performed. However, Keywords acid resistance; aldo-keto reductase; Helicobacter pylori; oxidoreductase Correspondence H. J. Windle, Institute of Molecular Medicine, Dublin Molecular Medicine Centre, Trinity College Dublin, St James Hospital, Dublin 8, Ireland Fax: +353 1 4542043 Tel: +353 1 8962211 E-mail: hjwindle@tcd.ie (Received 20 January 2008, revised 4 April 2008, accepted 9 April 2008) doi:10.1111/j.1742-4658.2008.06456.x Pyridine-linked oxidoreductase enzymes of Helicobacter pylori have been implicated in the pathogenesis of gastric disease. Previous studies in this laboratory examined a cinnamyl alcohol dehydrogenase that was capable of detoxifying a range of aromatic aldehydes. In the present work, we have extended these studies to identify and characterize an aldoketo reductase (AKR) enzyme present in H. pylori. The gene encoding this AKR was identified in the sequenced strain of H. pylori, 26695. The gene, referred to as HpAKR, was cloned and expressed in Escherichia coli as a His-tag fusion protein, and purified using nickel chelate chromatography. The gene product (HpAKR) has been assigned to the AKR13C1 family, although it differs in specificity from the two other known members of this family. The enzyme is a monomer with a molecular mass of approximately 39 kDa on SDS ⁄ PAGE. It reduces a range of aromatic aldehyde substrates with high catalytic efficiency, and exhibits dual cofactor specificity for both NADPH and NADH. HpAKR can function over a broad pH range (pH 4–9), and has a pH optimum of 5.5. It is inhibited by sodium valproate. Its substrate specificity complements that of the cinnamyl alcohol dehydrogenase activity in H. pylori, giving the organism the capacity to reduce a wide range of aldehydes. Generation of an HpAKR isogenic mutant of H. pylori demon- strated that HpAKR is required for growth under acidic conditions, sug- gesting an important role for this enzyme in adaptation to growth in the gastric mucosa. This AKR is a member of a hitherto little-studied class. Abbreviations AKR, aldoketo reductase; CAD, cinnamyl alcohol dehydrogenase; HpAKR, Helicobacter pylori aldoketo reductase. FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS 3041 disruption of the H. pylori genome upstream of the ORF for HpAKR gave rise to an acid-sensitive pheno- type. Thus, it was suggested that this enzyme may be involved in acid adaptation (polar effect), as it was located immediately 5¢ to the disruption point [3]. Our initial interest in alcohol-oxidizing ⁄ aldehyde- reducing enzymes of H. pylori stemmed from reports that these enzymes might contribute to the pathogenesis of H. pylori-associated damage to the gastric mucosa. It has been suggested that some of the H. pylori-induced damage to the gastric mucosa is mediated by toxic alde- hydes excreted from the cell. These excreted aldehydes were thought to react with proteins of the gastric mucosa, giving rise to inflammation, leading to gastritis [4–11]. This idea was supported by the lack of aldehyde dehydrogenase and aldehyde oxidase genes in the organ- ism. Thus, the ability of AKR to remove toxic aldehydes in the cell, by reduction to the corresponding alcohols, is of considerable interest. Previous research carried out in this laboratory detailed the characterization of a cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) from H. pylori [12]. This enzyme was the first CAD purified from a microbial source, and was shown to be similar to other characterized plant CAD enzymes in catalysing the reduction of aromatic aldehyde substrates. The CAD also displayed aldehyde detoxification by aldehyde dis- mutation. Thus, a pathway for detoxification of aro- matic aldehyde substrates was shown to exist in H. pylori [12]. However, the range of aldehydes reduced by CAD was limited, prompting us to exam- ine other aldehyde-reducing enzymes of this organism. The sequenced genome of H. pylori 26695 [13] har- bours a single putative AKR gene (Hp1193). The AKRs are a class of enzymes that typically catalyse the reduction of aldehydes and ketones to the corre- sponding alcohol product, and thus serve to remove potentially cytotoxic and mutagenic aldehydes from cells [14]. In view of the potential aldehyde detoxifying role of AKR within H. pylori, this study details the expres- sion, purification and characterization of a putative AKR from H. pylori 26695. HpAKR possesses low sequence identity to other characterized AKRs. The recombinant HpAKR exhibits specificity for aromatic aldehydes and ketones, with a high turnover, and is not involved in the metabolism of sugars or steroids. The recombinant enzyme demonstrates optimum activ- ity in an acidic environment at pH 5.5, which is similar to the pH of the mucous layer of the human stomach, where H. pylori resides. Furthermore, through inser- tional mutagenesis studies, we show that HpAKR is essential for growth at acid pH. Results Sequence analysis of HpAKR Sequence analysis of the HpAKR gene cloned in this study revealed the presence of an alanine residue at position 153 rather than the leucine residue docu- mented in the TIGR database (http://www.TIGR.org). The Hp1193 gene encodes a protein of 329 amino acids with an apparent molecular mass of 37 kDa. The sequence was submitted to the AKR superfamily data- base (http://www.med.upenn.edu/akr) for nomencla- ture assignment and was classified as AKR13C1. There are only two other members in this family, the AKR13A1 YacK protein from Schizosaccaro- myces pombe [15] and the AKR13B1 phenylacetalde- hyde dehydrogenase enzyme from Xylella fastidiosa [16]. A protein blast analysis revealed the highest sequence similarity with other putative AKRs and oxidoreductases from several different bacterial fami- lies. The greatest identity to HpAKR was observed for Yersinia frederiksenii ATCC 33641 (53% identity), Thermotoga maritima MSB8 (51% identity), Yersinia pestis KIM (51% identity), Azotobacter vinelandii (50% identity) and Escherichia coli CFT073 (50% identity). Overproduction of recombinant H. pylori AKR The putative HpAKR gene (Hp1193) was present in the sequenced strain of H. pylori 26695. Genomic DNA from this strain was used for subsequent amplifi- cation and cloning studies. HpAKR was cloned into E. coli DH5a, and the pET–Hp1193 construct contain- ing the inserted gene was transformed into E. coli BL21(DE3)plysS for overexpression. The His-tag pres- ent on the N-terminus of the expressed HpAKR facili- tated one-step affinity purification on a nickel-charged iminodiacetic acid column. The purity of each fraction was assessed using SDS ⁄ PAGE analysis. The gels were stained with Coomassie Brilliant Blue, and a protein species with an apparent molecular mass of 39 kDa (Fig. 1) was evident; this molecular mass compares favourably with that of 37 kDa obtained from the amino acid sequence. Additional minor bands of higher and lower molecular mass were apparent in the initial fractions eluting from the column. Substrate specificity and catalytic properties Kinetic parameters for HpAKR were determined using a wide range of aromatic and aliphatic aldehydes, H. pylori aldo-keto reductase D. Cornally et al. 3042 FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS dicarbonyls, ketones, sugars and steroids, as shown in Table 1. Optimum enzyme activity was observed for the dicarbonyl 9,10-phenanthrenequinone, with a K m value of 1 mm. The enzyme also possessed high activ- ity towards typical aldehyde substrates for AKRs, such as 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, and pyri- dine 2-aldehyde. NADPH could be replaced by NADH, although with some decrease in activity (Table 1). Unlike some aldose reductases and steroid dehydrogenases, HpAKR exhibited little or no activity with sugar or steroid substrates, including testosterone, oestrogen, cortisone, and progesterone. Stability and effects of pH HpAKR is an extremely stable enzyme, and it was able to withstand several freeze–thaw cycles with no loss of activity. The purified enzyme was stored at )20 °C for up to 6 months without significant loss of activity. Optimal reduction of 3-nitrobenzaldehyde by HpAKR was observed at pH 5.5 (Fig. 2). At pH 4, 50% activ- ity remained, whereas at pH 10, the activity was only 3% of that at pH 5.5. Inhibition studies Dithiothreitol inhibited HpAKR activity in a concen- tration-dependent manner, with maximal inhibition of 79% being observed with 20 mm dithiothreitol (Fig. 3). EDTA had no effect on HpAKR activity. Pyrazole, however, was a poor inhibitor (data not shown), with a 10% reduction in aldehyde reductase activity being observed at the highest concentration of pyrazole tested (0.8 mm). A characteristic of many aldehyde-reducing enzymes is their sensitivity to inhibition by sodium valproate. Sodium valproate was a reversible inhibitor of HpAKR. Kinetic analysis showed inhibition to be 45 Apparent molecular mass (kDa) 36 29 24 20 123 Fig. 1. SDS ⁄ PAGE of HpAKR: 15% SDS ⁄ PAGE indicating the pro- tein purity of recombinant HpAKR eluted from the nickel-charged iminodiacetic acid column. Lane 1 contains the nickel column wash. Lanes 2 and 3 show a single band for the Hp1193 protein with an approximate molecular mass of 39 kDa after staining with Coomas- sie Brilliant Blue. Table 1. AKR substrate specificity. Kinetic parameters of H. pylori AKR. The enzymatic activity was measured in the presence of 50 m M potassium phosphate buffer (pH 7.5) with 0.2 mM NADPH. All measurements made determined at 37 °C. All data are mean ± SEM (n = 3). NDA, no detectable activity. Substrate K m (mM) k cat (s –1 ) k cat ⁄ K m (mM –1 Æs –1 ) 3-Nitrobenzaldehyde 1.7 ± 0.2 399.3 ± 19.0 235.0 ± 11.0 4-Nitrobenzaldehyde 1.8 ± 0.3 416.9 ± 23.8 232.0 ± 35.0 Pyridine 2-aldehyde 1.7 ± 0.4 273.0 ± 3.4 160.0 ± 4.0 Pyridine 3-aldehyde 13.0 ± 1.2 111.3 ± 18.4 8.6 ± 1.6 Pyridine 4-aldehyde 3.6 ± 0.4 205.6 ± 7.0 56.9 ± 6.6 Benzaldehyde 1.9 ± 0.7 58.5 ± 6.0 30.8 ± 12.1 Succinic semialdehyde 10.0 ± 2.6 63.8 ± 5.7 6.4 ± 1.7 2-Methylbutyraldehyde 7.4 ± 2.0 90.7 ± 7.1 12.3 ± 3.1 Isatin 2.8 ± 0.1 122.4 ± 23.8 43.7 ± 8.6 Methylglyoxal 38.0 ± 9.4 261.5 ± 15.1 6.9 ± 1.7 Phenylglyoxal 2.0 ± 1.0 225.9 ± 26.7 113.0 ± 35.0 9,10-Phenanthrene- quinone 1.0 ± 0.1 274.3 ± 7.7 274.0 ± 31.0 NADH a 0.01 ± 0.004 17.8 ± 0.9 1776.0 ± 720.0 NADPH a 0.006 ± 0.001 65.6 ± 2.5 10 930 ± 1869.0 Cinnamaldehyde NDA – – Acetaldehyde NDA – – Coniferylaldehyde NDA – – Glyceraldehyde NDA – – D-Xylose NDA – – D-Glucose NDA – – D-Arabinose NDA – – Testosterone NDA – – Progesterone NDA – – Oestrogen NDA – – Cortisone NDA – – a Determined using 7 mM benzaldehyde. 2 3 4 5 6 7 8 9 10 0 250 500 750 1000 pH Velocity (µmol·min –1 ·mg –1 ) Fig. 2. The effects of pH on the activity of HpAKR. Initial rates of 3-nitrobenzaldehyde (3.2 m M) reduction were determined at the indicated pH values. The buffers used were as follows: pH 4–5, 50 m M sodium citrate; pH 6–8, 50 mM potassium phosphate; and pH 9–10, 50 m M glycine. D. Cornally et al. H. pylori aldo-keto reductase FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS 3043 essentially of a mixed type with respect to pyridine 2-aldehyde, as the apparent K m and V max values were altered in its presence (Fig. 4). However, the standard equation for such inhibition: v ¼ V max K m ½S ð1 þ ½I K i Þþð1 þ ½I K 0 i Þ where [S] and [I] are the pyridine 2-aldehyde and sodium valproate concentrations, respectively, was not applicable in this case. As shown in Fig. 4A, the curves at different sodium valproate concentrations demon- strate that both the K m and V max values are altered in the presence of the inhibitor. The graph of the reci- procal apparent 1 ⁄ V max values against sodium valpro- ate concentration was apparently linear (Fig. 4B), yielding a K i value of 220 ± 3 lm for the competitive element (K i ). However, this value should only be regarded as an approximation, as higher concentra- tions of sodium valproate appeared to cause the initial rate behaviour to depart from simple Michaelis– Menten kinetics. Variation of the apparent K m ⁄ V max values with sodium valproate concentration was clearly nonlinear (Fig. 4C). Thus, an inhibitor constant for the uncompetitive element of the inhibition (K¢ i ) could not be determined. Disruption of HpAKR by insertional mutagenesis and characterization of the isogenic mutant Insertional mutagenesis with a kanamycin cassette was performed to generate an isogenic mutant of H. pylori deficient in a functional AKR protein. PCR was used to confirm that the genomic copy of the gene had been disrupted by the kanamycin cassette, as demonstrated by the expected 1.5 kb increase in size of the PCR amplicon from genomic DNA of the transformed mutant (Fig. 5). A previous report in the literature had 0 2 4 6 8 10 12 14 16 18 20 0 50 100 150 200 DTT (m M) Velocity (µmol·min –1 ·mg –1 ) Fig. 3. Inhibition of HpAKR by dithiothreitol. The enzyme was preincubated at pH 7.5 at 37 °C with the indicated concentrations of dithiothreitol for 0 min (O) and 30 min (d) before determination of the activity towards 3-nitrobenzaldehyde. The data are presented as the mean of duplicate measurements. A B C V (µmol·min –1 ·mg –1 ) (1/V max ) Apparent(K m /V max ) Apparent Fig. 4. Inhibition of HpAKR by sodium valproate. The reductase activities towards a range of pyridine 2-aldehyde concentrations were determined at fixed sodium valproate concentrations. (A) The concentrations of sodium valproate were as follows: ,0mM; 4, 0.2 m M; , 0.4 mM; h, 0.8 mM; and d, 1.6 mM. Experimental values were fitted to the Michaelis–Menten equation by nonlinear regres- sion. (B) The dependence of the reciprocal apparent V max values on the concentration of sodium valproate. (C) The dependence of the apparent K m ⁄ V max values on the sodium valproate concentration. H. pylori aldo-keto reductase D. Cornally et al. 3044 FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS suggested a role for HpAKR in acid adaptation [3,17]. To test this hypothesis, both the isogenic mutant and the parental strain were grown in broth culture at dif- ferent pH values. No significant difference in growth rate between the mutant and wild-type was seen at pH 7.0 (Fig. 6A) or pH 6.0 (not shown). However, at pH 5.5 (Fig. 6B), the growth rate of the Hp AKR mutant was severely compromised beyond 10 h of growth, as compared to the wild-type. The growth rates of both the wild-type and the mutant were com- promised at pH 5.0 (not shown). The addition of urea (10 mm) to the medium at pH 5 and pH 5.5 resulted in a recovery in growth for both the wild-type and the mutant (data not shown). There was also a marked increase in the pH of the medium after 48 h of bacterial growth for both the wild-type and isogenic mutant. The pH rose from pH 5 or pH 5.5 to approximately pH 7.0. This pH change was not observed in the absence of urea. Discussion Sequence alignment HpAKR showed some sequence identity to several other putative bacterial AKRs. None with significant sequence identity (greater than 50%) to HpAKR has been characterized. On submission of the HpAKR sequence to the AKR superfamily homepage (http:// www.med.upenn.edu/akr), the enzyme was designated a new member of the AKR13 family and assigned the name AKR13C1. This family contains two other recognized members, AKR13A1 and AKR13B1. The substrate specificities of these other family members were, however, different from those of HpAKR. AKR13A1 demonstrated activity towards pyridine 2-aldehyde [15], but the catalytic efficiency (k cat ⁄ K m )of HpAKR for pyridine 2-aldehyde was six-fold higher than that of AKR13A1. The characterization of the second family member, AKR13B1, again was quite limited, as Michaelis constants were estimated for just two aldehyde substrates, glyceraldehyde and 2-nitro- benzaldehyde [16]. However, it is noted that AKR13B1 demonstrates activity towards glyceraldehyde, unlike the other two family members. 2.5 kb 2.0 kb 1.0 kb 12M Fig. 5. Agarose gel electrophoresis of pGEM:HpAKR::aphA-3 mutant: 1% agarose gel indicating the presence of the inserted aphA-3 cassette. Lane 1 shows a 1.5 kb increase in molecular mass in the pGEM:HpAKR construct, due to the presence of the inserted aphA-3 cassette amplified from H. pylori 1061. Lane 2 con- tains HpAKR, with an approximate size of 990 bp, amplified from H. pylori 1061. Lane M contains the DNA size marker. Fig. 6. Growth characteristics of H. pylori 1061 wild-type and AKR knockout mutant at various pH values. The growth characteristics of both H. pylori 1061 wild-type (h) and HpAKR knockout mutant ( ) in Brucella broth supplemented with 5% fetal bovine serum and Dent supplement at either pH 7.0 (A) or pH 5.5 (B). The results are shown as the mean of duplicate determinations. D. Cornally et al. H. pylori aldo-keto reductase FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS 3045 Substrate specificity Generally, AKRs have been associated with detoxifica- tion of a broad range of aldehyde substrates. In this context, HpAKR demonstrated a high turnover towards a wide range of aldehyde substrates. The K m values were generally within the 1–10 mm range, with the exception of methylglyoxal (38 mm). The enzyme’s affinity towards these various aromatic aldehyde sub- strates is clearly indicated by the estimated kinetic con- stants. The highest k cat values were obtained for the nitrobenzaldehydes, which is typical of microbial AKRs [18]. However, unlike the AKR from Digi- talis purpurea and xylose reductase from Candida par- apsilosis [19,20], HpAKR showed no significant activity towards sugar or steroid substrates, thus indi- cating it was not a member of the AKR subgroups aldose reductase and hydroxysteroid dehydrogenase. In this respect, the enzyme resembles AKR7A5 and AKR1C19 [21,22]. In terms of aldehyde toxicity, it is clear that this enzyme, due to its high turnover rate, will efficiently detoxify aromatic aldehydes that enter the cell or are produced endogenously. Thus, it is unlikely that such aldehydes would be exported from H. pylori and inter- act with the gastric mucosa. This does not rule out the possibility that other aldehydes might be exported from the cell. This will only be evident when a full profile of all aldehyde-oxidizing activities in the cell is known. Another unusual property associated with HpAKR was its ability to display dual coenzyme specificity for NADH and NADPH, although with a preference for NADPH. This would place it in the EC 1.1.1.71 alde- hyde reductase class. In this respect, it is similar to the aflatoxin-metabolizing aldehyde reductase, AKR7A5, AKR1C19, and the thermostable alcohol dehydroge- nase from E. coli [21–24]. In contrast, some yeast [20,25,26] and plant [19,27] AKRs do not possess such dual coenzyme specificity. The other two members of this family, AKR13A1 and AKR13B1, have been shown to be specific for NADPH [15,16]. pH activity profiles The optimum pH for HpAKR activity, with 3-nitro- benzaldehyde as the substrate and NADPH as the cofactor, was around pH 5.5, but it is apparent that the enzyme can function over a broad pH range (pH 4–9). The pH activity profile is similar to that for AKR7A5 and the AKR from Saccharomyces cerevisiae [21,26,28]. However, several other members of the AKR family have been reported only to function over a narrow range, from pH 6 to pH 8. These include xylose reductase from C. parapsilosis [20], AKR1C19 [22], the thermostable alcohol dehydrogenase from E. coli [23], benzil reductase from Bacillus cereus [29], pyridoxal reductase from Sc. pombe [30], and aldehyde reductase from pig liver [31]. Inhibition studies The inhibition of HpAKR activity by dithiothreitol is most unusual for the AKR family of enzymes. It might suggest that two of the three cysteine residues present on the enzyme may be involved in the formation of a disulfide bridge, which is necessary for HpAKR activ- ity. The lack of inhibition by EDTA (not shown) sug- gests that an accessible divalent cation is not required for HpAKR activity. The alcohol dehydrogenase inhibitor pyrazole was shown to be a poor inhibitor of HpAKR, which is similar to the behaviour of the mouse liver morphine-6-dehydrogenase [32]. Sodium valproate is a known potent AKR inhibitor [21,23,26,32]. Inhibition by sodium valproate has been used to distinguish aldehyde reductases from aldose reductases, although not all aldehyde reductases are sensitive to inhibition by this compound [33]. The kinetic behaviour of HpAKR in the presence of sodium valproate was complex. Similar behaviour has been reported by others [34] for the AKR from sheep liver; the authors ascribed this to the formation of enzyme–valproate and enzyme–NADPH–valproate complexes in the inhibitory process. Such behaviour precludes the determination of the K i values. Construction of isogenic mutant Previous research suggested that the disruption of the H. pylori genome upstream of the ORF for HpAKR resulted in the bacteria exhibiting acid sensitivity. Interestingly, these workers suggested that the enzyme was required when exposure to acid conditions was chronic [3]. Here, we have characterized this acid sensitivity and tested the hypothesis that an intact copy of HpAKR is essential for growth under acidic conditions. An iso- genic mutant of H. pylori lacking the AKR protein was unable to grow at pH 5.5, whereas the parental strain at the same pH repeatedly grew, albeit at a slower rate than that observed under neutral condi- tions. This result was robust and was repeated several times. Both the wild-type and mutant failed to grow below pH 5.5. Deletion of HpAKR had no effect on growth at either pH 7.0 or pH 6.0. The addition of urea to the medium at pH 5.0 and pH 5.5 resulted in an increase in growth for H. pylori aldo-keto reductase D. Cornally et al. 3046 FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS both the parental strain and the mutant. There was also a marked increase in pH after 48 h of growth from pH 5 or pH 5.5 to approximately pH 7.0. It would seem that the increase in growth rate observed at this low pH is due to the rise in pH generated as a result of urease activity. No such pH increase was seen in the absence of urea. Similar findings have been reported by Clyne et al. [35], who ascribed an increase in the pH to the production of ammonia as a result of urease activity. To conclude, we have shown the Hp1193 product (HpAKR) to be an active AKR that differs in speci- ficity from the two other members of the AKR13C1 family. The assigned putative role of Hp1193 can now be confirmed on the basis of the kinetic analysis undertaken in this study. The recombinant enzyme possesses low sequence identity and has an unusually high turnover rate in comparison to other enzymes within its class. It exhibited a broad substrate specific- ity profile, with optimum activity being demonstrated at approximately pH 5.5. The AKR is the second aldehyde-reducing enzyme described in H. pylori, demonstrating that aldehydes produced by this organism can be efficiently reduced, and this may have implications for the theory that aldehyde accumulation contributes to the pathogenicity of H. pylori [4–11]. HpAKR is required for growth under acidic condi- tions, and as such this protein may represent a thera- peutic target. As the presence of urea rescues this mutant from acid intolerance, the AKR appears to contribute to a process that supports growth under acid conditions where urea is lacking. This may be significant in long-term colonization of the gastric mucosa, and it appears that a few other gene products may also contribute [3]. Further work will be necessary to elucidate the role of this activity in the colonisation of the gastric mucosa by H. pylori. Experimental procedures Cloning and characterization of the recombinant HpAKR Materials Restriction enzymes were from New England Biolabs (Herts, UK). Taq-High Fidelity polymerase was from Roche (Basel, Switzerland). T4 DNA ligase was from Invi- trogen (Paisley, UK). Iminodiacetic acid–Sepharose 6B fast flow, NADH, NADPH, alcohol and aldehyde substrates and isopropyl thio-b-d-galactoside were obtained from Sigma Aldrich (Dublin, Ireland). Bacterial strains and plasmids Genomic DN A fro m H. pylori 26695 was used to amplify Hp1193 by PCR. The pET 16b vector (Novagen, Madison, WI, USA) was used to clone and overexpress Hp1193 in E. coli BL21(DE3)plysS. E. coli was grown at 37 ° C in LB medium supplemented with ampicillin (100 lgÆ mL )1 ) and chloram- phenicol (34 lgÆmL )1 ) to select for the desired constructs. Cloning methods All DNA manipulations were performed under standard conditions as described in [36]. The AKR gene was amplified by PCR using genomic DNA from H. pylori 26695 as the template, and the oligonucleotides 5¢-CGC CAT ATG CAA CAG CGT CATT-3¢ and 5¢-CGC GGA TCC TTG ATT CAC CAT TTC AT-3¢ as the forward and reverse primers, respectively. These primers were designed to introduce an NdeI site (underlined in the forward primer) and a BamHI site (underlined in the reverse primer). The amplified PCR product, containing Hp1193, was cloned into the pET 16b vector (Novagen; all pET vectors are derived from plasmid pBR322). The resulting construct was named pET–Hp1193. The construct was sequenced in both directions (DNA sequencing facility, University of Cambridge, UK). Purification of the Hp1193 gene product Overproduction of the recombinant HpAKR was achieved in E. coli BL21(DE3)plysS. Cells harbouring pET–Hp1193 were grown to an D 600 nm of 0.6, in LB medium containing ampicillin (100 lgÆmL )1 ) and chloramphenicol (34 lgÆmL )1 ). Production of HpAKR was initiated by addition of isopropyl thio-b-d-galactoside to a final concentration of 1 mm, and this was followed by incubation at room temperature, to minimize inclusion body formation. After 14 h, the cells were harvested by centrifugation at 5000 g for 30 min at 4 °C. For protein purification, the cells from a 1 L culture were resus- pended in 50 mL of binding buffer (5 mm imidazole, 0.5 m NaCl, 20 mm Tris ⁄ HCl, pH 7.9) and sonicated on ice for 3 · 4 min (Soniprep 150, Sanyo, Loughborough, UK). The resulting cell lysate was centrifuged at 5000 g for 1 h at 4 °C, and the supernatant was filtered (0.45 lm) prior to loading onto a nickel-charged iminodiacetic acid column. Unbound material was eluted using 10 column volumes of binding buf- fer (10 m m imidazole, 0.5 m NaCl, 20 mm Tris ⁄ HCl, pH 7.9) and six column volumes of wash buffer (60 mm imidazole, 0.5 m NaCl, 20 mm Tris ⁄ HCl, pH 7.9). The recombinant AKR protein was then eluted over seven column volumes with elution buffer (300 mm imidazole, 0.5 m NaCl, 20 mm Tris ⁄ HCl, pH 7.9). SDS ⁄ PAGE was performed essentially as described in [37] to monitor the purity of each fraction. Proteins were D. Cornally et al. H. pylori aldo-keto reductase FEBS Journal 275 (2008) 3041–3050 ª 2008 The Authors Journal compilation ª 2008 FEBS 3047 visualized by Coomassie blue staining. The purified protein was dialysed against 50 mm potassium phosphate buffer (pH 7.5) containing 50 lm EDTA, with two buffer changes. The enzyme was concentrated using Centricon ultrafiltra- tion tubes, and stored at )20 °C. The protein concentration was determined by the Bradford method [38], using BSA as the protein standard. Enzyme assay The kinetic parameters for aldehyde reduction were esti- mated using a spectrophotometric assay at 37 °C using an Agilent 8453 diode array spectrophotometer. The purified enzyme was assayed for the reduction of aldehydes. Activities towards different aldehydes were assayed in reaction mix- tures (2 mL) containing 50 mm potassium phosphate buffer (pH 7.5) with 0.2 mm NADPH. The decrease in NADPH absorbance at 340 nm was followed. The molar extinction coefficient (e) used (pH 7.5) was: e 340 = 6.22 mm )1 Æcm )1 for NADPH. Steady-state parameters were determined by fitting initial rates to the Michaelis–Menten equation using the enz- fitter program (Elsevier Biosoft, Cambridge, UK), and data for the inhibition by sodium valproate were analysed by non- linear regression using the program maccurvefit (Kevin Ra- ner Software, Mt Waverly, Victoria, Australia). Construction of H. pylori AKR mutant by insertional mutagenesis Bacterial strains and plasmids H. pylori strains 26695 and 1061 were provided by A. Van Vliet and J. Kusters (Erasmus MC University Medi- cal Centre, Rotterdam, the Netherlands). Strains of H. pylori (26695 and 1061) were grown on Columbia agar (Oxoid, Basingstoke, UK) plates containing defibrinated horse blood (7%, v ⁄ v) in a microaerobic (Anoxomat) atmosphere at 37 °C. E. coli strains were routinely grown in LB broth and on LB agar. The antibiotics used for selection purposes were ampicillin (50 lgÆmL )1 ) and kanamycin (20 lgÆmL )1 ). DNA manipulation Unless stated otherwise, all DNA manipulation techniques were performed using standard procedures [39]. Transfor- mation of the E. coli cloning host (DH5a) was performed using standard methods. Natural transformation of H. pylori with plasmid constructs was performed as described in [40]. All oligonucleotide primers were obtained from Sigma-Genosys (UK). Construction of the AKR mutant The purified PCR-amplified AKR gene (Hp1193) was ligated into the cloning vector pGEM-T Easy (Promega Southampton, UK). The primers used to amplify the gene were: forward, 5¢-ATG CAA CAG CGT CAT T-3¢; and reverse, 5¢-TTA TTG ATT CAC CAT TTC AT-3¢. A 1.5 kb PCR product from plasmid pJMK30 containing a gene encoding resistance to kanamycin was amplified using the universal sequencing primers M13 and cloned into the unique XcmI site within HpAKR to yield pGEM:HpAKR::aphA-3. This construct was digested with PsiI (generating two frag- ments) to determine the orientation of the aphA-3 cassette. PCR and DNA sequencing were used to confirm the disrup- tion of the gene. The appropriate construct was used for nat- ural transformation of H. pylori 1061, essentially as described in [40]. H. pylori genomic DNA was purified using the Puregene DNA Isolation kit (Gentra Systems, Minneap- olis, MN, USA), and PCR was used to confirm the presence of the disrupted copy of genomic HpAKR. Broth culture For liquid culture, strains were grown in Brucella broth (GIBCO BRL, Life Technologies, Paisley, UK) supple- mented with 5% fetal bovine serum. To ensure that all strains were in the same growth phase, the bacteria were first grown to an D 600 nm of approximately 1, and then diluted in this medium so that an D 600 nm of 0.05 was obtained. Cultures were grown in 10 cm 2 cell culture flasks (Nunclon, Roskilde, Denmark), in a micro-aerobic environment, at 120 r.p.m. An acidic environment was created using Brucella broth, which was adjusted to the desired pH using HCl after the addition of fetal bovine serum and Dent supplement and subsequently filter sterilized. For the isogenic mutant, the medium was supplemented with kanamycin (20 lgÆmL )1 ). 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There was also a marked increase in pH after 48 h of growth from pH 5 or pH 5.5 to approximately pH 7.0. It would seem that the increase in growth rate observed at this