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Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding
Life Sciences 85 (2009) 303–308
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding☆ Takeshi Miura a,b,⁎, Toru Nishinaka a, Tomoyuki Terada a a b
Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan Department of Clinical Research, National Hospital Organization Hakodate Hospital, 18-16 Kawahara-cho, Hakodate, Hokkaido 041-8512, Japan
a r t i c l e
i n f o
Article history: Received 13 November 2008 Accepted 15 June 2009 Keywords: Short-chain dehydrogenase/reductase Anthracycline Chimera CBR Harr plot Swapping
a b s t r a c t Aims: Monomeric carbonyl reductase 1 (CBR1) and 3 (CBR3) are members of the short-chain dehydrogenase/ reductase superfamily, and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR3 exhibits a higher Km value toward NADPH and more limited carbonyl reductase activities than CBR1, although they are highly homologous to each other in amino acid sequence levels. In the present study, we investigated the origin of the different properties of the enzymes by analyses using several chimeric enzymes. Main Methods: Harr-plot analysis of the amino acid sequences was conducted and as a result, two low-identity regions between human CBR1 and CBR3 were found: these were designated as the N-terminal low-identity region (LirN) and the C-terminal low-identity region (LirC; the substrate-binding region). We genetically constructed chimeric enzymes while focusing on these regions. Key findings: Chimeric CBR1 possessing LirN of CBR3 (CBR1LirN3) exhibited CBR1-like activities but a low coenzyme affinity probably due to a structural alteration in a micro domain, whereas chimeric CBR1 including LirC of CBR3 (CBR1LirC3) was enzymatically similar to CBR3. Furthermore, CBR3LirC1 was similar to CBR1 in both enzymatic activities and coenzyme binding. Significance: These results suggested that LirC, i.e., the substrate-binding loop region, is the origin of the difference between human CBR1 and CBR3 in both catalytic and coenzyme-binding properties. © 2009 Elsevier Inc. All rights reserved.
Introduction The short-chain dehydrogenase/reductase (SDR) superfamily is a large group of enzymes with NAD(P)(H)-dependent oxidoreductase activities including hydroxysteroid dehydrogenase, prostaglandin (PG) oxidoreductase, retinal reductase/dehydrogenase, and carbonyl reductase (Hoffmann and Maser 2007; Oppermann 2007; Matsunaga et al. 2006; Forrest and Gonzalez 2000). In general, a low-sequence identity (b30%) is observed among the SDR enzymes, and the homologous region is restricted to the residues that are involved in the binding of coenzymes (the Rossmann-fold consensus sequence) and catalysis (Hoffmann and Maser 2007). Monomeric carbonyl reductases (CBRs) are members of the SDR superfamily. Two CBRs are found in humans: carbonyl reductase 1 (CBR1) (Wermuth et al. 1988) and carbonyl reductase 3 (CBR3) (Watanabe et al. 1998). The enzymes share high ☆ Part of this study was presented at the 8th International ISSX (the International Society for the Study of Xenobiotics) meeting on 9–12, Oct. 2007 (Sendai, Japan). Dr. E. Maser and his colleagues presented, on Jul. 8–12 2008, at the 14th International Meeting on Enzymology and Molecular Biology of Carbonyl Metabolism (Kranjska gora, Slovenia) regarding the origin of the difference between CBR1 and CBR3, and reached at essentially the same conclusion that we did. ⁎ Corresponding author. 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan. Tel.: +81 721 24 9983; fax: +81 721 24 9890. E-mail address: [email protected] (T. Miura). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.06.005
identity (72%) in their amino acid sequences. Both the N-terminal Rossmann-fold consensus sequence (GxxxGxG; 12–18 at amino acid residue) and the catalytic triad (S-YxxxK; Ser139, Tyr193, and Lys197) in the center and C-terminus of the enzymes are completely conserved. CBR1 is a reductase for endogenous and xenobiotic compounds using NADPH as a coenzyme, and has been known as a PG 9-keto reductase which is one of the physiological regulators of PG metabolism (Wermuth 1981), and also as a metabolizing enzyme for anthracycline anti-cancer drugs such as daunorubicin and doxorubicin (Kassner et al. 2008; Gavelová et al. 2008; Ax et al. 2000; Forrest et al. 1991). The metabolites of anthracycline (13-hydroxy anthracycline) that are inactivated by CBR1 are believed to induce congestive heart failure, which is sometimes lethal (Olson et al. 2003; Forrest et al. 2000). Furthermore, increased expression of CBR1 has caused the resistance to the drugs (Plebuch et al. 2007; Gonzalez et al. 1995). Therefore, the development of selective inhibitors to CBR1 is advisable for more efficient anthracycline dosage (Carlquist et al. 2008). On the other hand, the physiological roles of CBR3 remain unknown. Although genetic variants of CBR3 may correlate the pharmacokinetics of doxorubicin (Fan et al. 2008; Blanco et al. 2008), CBR3 is not an efficient reductase that reduces doxorubicin to 13-hydroxy doxorubicin (Kassner et al. 2008). In our recent investigations (Miura et al. 2008, 2009a,b), human CBR3 and the orthologues of Chinese hamsters and rats exhibited limited carbonyl reductase activities for the test substrates. Furthermore,
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the Km value of CBR3 toward NADPH was higher than that of CBR1. These observations suggested that CBR1 and CBR3 play different physiological roles although they are highly homologous. The clarification of the origin of the enzymatic differences between CBR1 and CBR3 is useful not only in terms of fostering a deeper understanding of both the enzymatic mechanism and physiological roles of the enzymes but also for the development of CBR1-selective inhibitors. Thus, we bioinformatically analyzed their amino acid sequences and identified two low-identity regions. In the present study, the contribution of these low-identity regions to the enzymatic differences between the enzymes was investigated by conducting analyses of several chimeric enzymes that may become powerful tools for the development of selective inhibitors. Materials and methods Materials All reagents in this study were of the highest grade that is commercially available. Construction of bacterial-expression vectors for chimeric CBR Bacterial-expression vectors for CBR1 and CBR3 (pET-hCBR1 and pET-hCBR3, respectively) were previously described (Miura et al. 2008). Bacterial-expression vectors for chimeric proteins were designated as indicated in Fig. 2. The N-terminal region of CBR3 cDNA was amplified by polymerase chain reaction (PCR) using hmhCBR1f03(NdeI) (5′-cgc ata tgt tcc gcg cgc cc-3′; bold, NdeI-recognition site) and mthCBR3r03 (5′-tcg ggt acc aaa aaa att tgt ctt cag tgt ca-3′; bold, KpnI-recognition sequence) as primers, and excised with NdeI and KpnI. The DNA fragment was inserted into the NdeI–KpnI site in pET-hCBR1. The resulting vector was termed pET-hCBR3/1. Similarly, for the construction of pEThCBR1/3, the C-terminal region of CBR3 cDNA was amplified by PCR using mthCBR3f03 (5′-ttt ggt acc aga aac atg tgc aac gag tta-3′; bold, the KpnI-recognition sequence) and hmhCBR3r03(EcoRI) (5′-acg aat tca gga caa ggt aca aaa tgg ggc-3′; bold, the EcoRI-recognition site) as primers, excised with KpnI and EcoRI, and inserted into the KpnI– EcoRI site in pET-hCBR1. The constructions of pET-hCBR1LirN3, pET-hCBR1LirC3, and pEThCBR3LirC1 were performed with the In-fusion PCR cloning kit (Clontech Laboratories, Inc., Palo Alto, CA, USA): DNA fragments of the chimeric region (LirN or LirC) in hCBR1 or hCBR3 were reacted with the products of inverted tail-to-tail PCR using pET-hCBR1 or pET-hCBR3 as templates. The primers were as follows; mthCBR1f12 (5′-agt gag acc atc act gag gag gag ctg-3′), mthCBR1r13 (5′-aga tac gtt cac cac tct ccc ttg ggg3′), mthCBR3f07 (5′-gtg gtg aac gta tct agt ttg cag tgt tt agg-3′), and mthCBR3r07 (5′-agt gat ggt ctc act gtg gaa cct ttc ctg-3′) for pEThCBR1LirN3; mthCBR1f05 (5′-acc ctg tgt act tgg ccc tt-3′), mthCBR1r06 (5′-gca gca agg cat tca gga gga tc-3′), mthCBR3f05 (5′-ctg aat gcc tgc tgc cca gga cca gtg ccg-3′), and mthCBR3r05 (5′-caa gta cac agg ggt ctc agc ccc ctg ctc-3′) for pET-hCBR1LirC3; mthCBR3f04 (5′-acc cct gtc tac ttg gcc ctc-3′), mthCBR3r04 (5′-gca gca cgc att cac cag aat c-3′), mthCBR1f11 (5′-gtg aat gcg tgc tgc cca ggg tgg gtg aga-3′), and mthCR1r12 (5′-caa gta gac agg ggt ctc tgc acc ttc ttc-3′) for pET-hCBR3LirC1. pET-hCBR1LirNC3 was constructed by the ligation of the NdeI/KpnI fragment of pET-hCBR1LirN3 to the NdeI/KpnI site of pEThCBR1LirC3. Bacterial expression and purification of recombinant wild-type and chimeric enzymes
When the absorbance of the culture at 600 nm was 0.5–0.8, isopropyl-βD-thiogalactopyranoside (IPTG) was added to the medium at a concentration of 0.2 mM, and the cells were cultured for an additional 3–4 h. The bacterial cells were then harvested, washed with 20 mM sodiumphosphate buffer, pH 7.5 (buffer A), resuspended with 30 ml of buffer A, and then sonicated. After centrifugation of the crude extract at 17,000 ×g for 30 min, the supernatant was subjected to purification using Ni++ chelating affinity column chromatography (bed volume, 1 ml). After equilibrating the column with wash buffer 1 (50 mM potassium-phosphate buffer, pH 8.0, with 300 mM KCl and 5 mM imidazole), the supernatant was loaded. The column was washed with 6 ml of wash buffer 1 and then 6 ml of wash buffer 2 (50 mM potassium-phosphate buffer, pH 8.0, with 300 mM KCl and 10 mM imidazole). The protein adsorbed to the affinity resin was eluted with elution buffer (50 mM potassium-phosphate buffer, pH 8.0, with 300 mM KCl and 250 mM imidazole). The eluted protein was gelfiltrated with Bio-Gel P-6 (Bio-Rad Laboratories, Hercules, CA, USA) preequilibrated with buffer A, and then dithiothreitol (DTT) was added at a final concentration of 5 mM. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard (Bradford 1976). The purified CBR proteins exhibited a single band on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue G-250 (CBB) staining. Determination of enzymatic activity The reaction mixture for the reductase activity consisted of 100 mM sodium-phosphate buffer, pH 6.5,130 µM NADPH, and the substrate. The reaction was initiated by the addition of an appropriate amount of enzyme. The enzymatic activity was monitored by measuring the decrease in absorbance at 340 nm depending on the conversion of NADP (H) at 25 °C. Five concentrations of each substrate were assayed at the minimum, and each concentration was measured at least three times. These reactions were performed at a total volume of 1 ml. Menadione and 4-benzoylpyridine stock solutions were prepared in methanol. The final concentration of methanol in the reaction was not more than 10% (v/v). The apparent Km and kcat values were determined by fitting the initial velocities to the Michaelis–Menten equation with SigmaPlot (Systat Software, Inc., Richmond, CA, USA) based on a molar extinction coefficient for NADPH of 6.22 × 103 M− 1 cm− 1. Each kinetic constant was the average of three measurements. Coenzyme binding To determine coenzyme dissociation constants, we measured the changes in the intrinsic enzyme fluorescence on the incremental addition of coenzymes on a spectrofluorophotometer (RF-5300PC; Shimadzu Co. Ltd., Kyoto, Japan). To ensure that the volume of coenzyme added did not exceed 3% of the total volume, three stock solutions of the coenzyme were prepared: 13 mM, 6.5 mM, and 1.3 mM. A 4 × 10-mm cuvette was used, and each 3 ml sample contained 60 µg of proteins in 100 mM sodium-phosphate buffer, pH 6.5 at 25 °C. The samples were excited at 290 nm and the fluorescence emission was scanned at 335 nm. Hill's equation was fitted to the fluorescence decreases that were dependent on coenzyme binding in order to estimate the corresponding dissociation constants for the wild-type and chimeric enzymes using SigmaPlot. Kd values were obtained from three experiments performed with at least two independent protein preparations. Results Identification of low-identity regions between CBR1 and CBR3
The bacterial cells [E. coli, BL21(DE3)pLysE] transformed by various bacterial-expression vectors were precultured overnight in Luria (L)-broth containing 25 µg/ml kanamycin at 37 °C with vigorous shaking, and then further cultured in 25 volumes of L-broth under the same conditions.
Regarding the orthologues of humans, Chinese hamsters, and rats, CBR3 exhibits extremely lower catalytic carbonyl reductase activity than CBR1, although both enzymes share high identity (70–75%) in
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Fig. 2. Structures of chimeric enzymes. The amino acid sequences of CBR1 and CBR3 were shown as opened and closed boxes, respectively.
both LirN and LirC) includes the enzymatic determinant of specific properties of CBRs. Specific properties of CBRs were dependent on the substrate-binding loop region
Fig. 1. (A) Harr-plot analysis for human CBR1 and CBR3 in their amino acid sequences. Harr-plot analysis was performed under the following conditions: unit size to compare, 5; dot plot score, 3.00. Two low-identity regions were termed the N-terminal lowidentity region (LirN; 140–159 at amino acid residue) and the C-terminal low-identity region (LirC; 230–244 at amino acid residue), respectively. (B) Sequence alignment of LirN between CBR1 and CBR3. (C) Sequence alignment of LirC between CBR1 and CBR3.
their amino acid sequences. To investigate the origin of the enzymatic difference, Harr-plot analysis (Harr et al. 1982; Staden 1982) was used to compare the amino acid sequences (Fig. 1; performed by GENETYX software; Genetyx Co. Ltd., Tokyo, Japan). The analysis is a twodimensional graphical homology search in nucleotide and amino acid sequences. Harr-plot analysis between CBR1 and CBR3 revealed a discontinuous-line pattern, and two low-identity regions were found as opening spaces in the line (Fig. 1A). One was the region near catalytic Ser-139 (140–159 at amino acid residue; the N-terminal lowidentity region, LirN), and formed two α helix structures, which are αEF1 and αEF2 as considered from the crystal structures of CBR1 (Protein Data Bank No.: 1WMA) and CBR3 (Protein Data Bank No.: 2HRB) (Fig. 1B). The other was the substrate-binding loop region (230–244 at amino acid residue) (Fig. 1C), which was designated as the C-terminal low-identity region (LirC). The identification of these two low-identity regions in CBR prompted us to study the involvement of these regions in the enzymatic properties by characterizing the chimeric enzymes between CBR1 and CBR3. Contribution of the C-terminal region of CBRs for enzymatic properties First, we examined whether the two regions in CBR1 and CBR3 can affect their carbonyl reductase activities. Chimeric enzymes, CBR3/1 and CBR1/3, contain both LirN and LirC of CBR1 and CBR3, respectively (Fig. 2). The kinetic constants of these enzymes toward test substrates (menadione and 4-benzoylpyridine) for carbonyl reductase activity were determined (Table 1). CBR3/1 and CBR1/3 exhibited kinetic constants similar to CBR1 and CBR3, respectively, suggesting that the C-terminal region of CBRs (118–277 at amino acid residue; containing
The above-mentioned results implied the possibility that either LirN or LirC affects the specific properties of CBR1 and CBR3. Therefore, we characterized the chimeric enzymes more precisely (Table 1). The chimeric enzyme of CBR1 containing LirN of CBR3 (CBR1LirN3) exhibited almost CBR1-like kinetic constants, although its catalytic efficiencies were low as compared to those of CBR1. On the other hand, CBR1LirC3 and CBR1LirNC3 showed low catalytic efficiencies, which were similar to CBR3. Moreover, the exchange of LirC in CBR3 for that of CBR1 (CBR3LirC1) increased the kcat and catalytic efficiencies of the enzymes. Similarly, the Km values toward NADPH were dependent on LirC, not LirN. These results suggested that specific properties for catalysis of CBR1 and CBR3 are dependent on LirC, i.e., the substratebinding loop region. Coenzyme binding properties of wild-type and chimeric enzymes The Km values of CBR3 toward NADPH were reported to be higher values than those of CBR1 (an order of 1 µM) (Miura et al. 2008; Lakhman et al. 2005; Usami et al. 2001; Bohren et al. 1987), and we obtained reproducible results (Table 1). We used chimeric enzymes to determine the critical region(s) where such a coenzyme-binding difference between CBR1 and CBR3 exists. The binding of the enzymes to NADPH was measured by the decrease in the intrinsic enzyme fluorescence on the incremental addition of NADPH. The fluorescence spectra of CBR1 and CBR3 without NADPH are shown in Fig. 3A. Table 1 Menadione CBR1 CBR3/1 CBR1LirN3 CBR1LirC3 CBR1LirNC3 CBR3LirC1 CBR1/3 CBR3
4-benzoylpyridine
NADPH
Km
kcat
kcat/Km
Km
kcat
kcat/Km
Km
Kd
30 49 34 49 42 45 65 54
410 110 41 0.079 0.053 59 0.88 0.32
14 2.2 1.2 0.0016 0.0013 1.3 0.014 0.0059
360 400 380 470 420 450 360 300
400 270 140 0.37 0.20 64 1.2 0.70
1.1 0.68 0.37 0.00079 0.00048 0.14 0.0033 0.0023
6.2 5.3 7.4 16 19 5.8 32 26
1.2 0.21 5.7 7.1 7.3 0.66 21 7.5
Km and Kd values were shown as µM. kcat values were shown as min− 1. To investigate kinetic constants toward coenzymes, the concentration of menadione was held at 200 µM. Standard deviation of each value was within 25%.
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Kd values that were distinct from those of CBR1: the Kd values of these chimeric enzymes were higher than those of CBR1. Therefore, the Kd value of CBR toward NADPH was suggested to be dependent on LirC. The Kd values of wild-type and chimeric enzymes toward NADPH, which are lower as compared to the Km values, might be due to insufficient saturation of the substrate, menadione. Discussion
Fig. 3. Fluorescence titrations of CBR for NADPH. (A) Fluorescence spectra of CBR1 and CBR3 with the excitation at 290 nm are shown. The intensities of fluorescence at 335 nm at each protein were supposed to be 1.0 as a standard in order to observe the differences in the fluorescence peak. Solid line, CBR1; broken line, CBR3. (B, C) Typical fitting curves are indicated. The decreases in relative fluorescence (ex. 290 nm/em. 335 nm) of CBR1 (B) and CBR3 (C) fitted to the curves of Hill's equation. The Hill plot was in each graph to consider the degree of linear regression.
The wavelength at the fluorescence peak of CBR3 slightly differed from that of CBR1.With regard to the wavelength to observe the intrinsic fluorescence of CBR1 and CBR3, the Kd values estimated by the fluorescence decrease at 335 nm on the addition of NADPH were essentially the same as the values estimated by the fluorescence decrease at 325, 330, 340, and 345 nm (data not shown). Therefore, the data at 335 nm were used in the present study to measure the Kd values. The Kd value of CBR1 toward NADPH was in good agreement with a previous report (Bohren et al. 1987). The Kd value of CBR1 was clearly lower than that of CBR3. Moreover, CBR3/1 and CBR3LirC1 exhibited Kd values similar to CBR1. On the other hand, chimeric enzymes that have the LirC of CBR3 (CBR1/3, CBR1LirC3, and CBR1LirNC3) showed
CBRs are members of the SDR superfamily and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR1 and CBR3 share high identity (70–75%) in their amino acid sequences (Hoffmann and Maser 2007; Oppermann 2007; Matsunaga et al. 2006; Forrest and Gonzalez 2000). CBR1 has been well characterized from the views of both drug metabolism and enzymology. A recent study (Kassner et al. 2008) revealed that in humans, CBR1 is a dominant enzyme that metabolizes doxorubicin, an anticancer drug, in the livers whereas CBR3 does not contribute to the inactivation of the drug. Furthermore, in our investigations (Miura et al. 2008, 2009a,b; Terada et al. 2001, 2003), CBR3 exhibited lower carbonyl reductase activities toward test substrates than CBR1 in humans, Chinese hamsters, and rats. These results suggested that the limited carbonyl reductase activity of CBR3 is a common property among animal species. Moreover, although both the coenzymebinding motif (the Rossmann-fold consensus sequence, GxxxGxG in the N-terminus of the proteins) and the “NADPH-specificity motif KAR” (Hoffmann and Maser 2007) near the Rossmann-fold consensus sequence are highly conserved between CBR1 and CBR3, the Km value of CBR3 toward NADPH was suggested to be highly distinct from that of CBR1. In the present study, in order to clarify the origin of these differences, we bioinformatically searched low-identity regions between CBR1 and CBR3. Based on the result (Fig. 1), we genetically constructed and characterized several chimeric enzymes, and finally, identified the substrate-binding loop region in CBR1 and CBR3 as the region that is responsible for the specific properties of the two enzymes. Substrates binding with the SDR enzymes are expected to be followed by both the swinging and accommodating of the loop without an apparent conformational change of the loop structure (Tanaka et al. 1996), suggesting that unique properties of CBR1 and CBR3 are mainly contributed by the structural differences between the loop structures. Low-identity regions that were investigated in the present study were rendered by the result of Harr-plot analysis (Fig. 1). The analysis is a graphical homology search (Harr et al. 1982; Staden 1982). Since the result is graphically provided, the analysis simplifies the evaluation of the result from homology search; therefore, the analysis has the power to appeal the existence of local regions that exhibit high or low identity for the human eyes, which is usually a weak point of the simple homology search. Thus, as we easily identified low-identity regions as targets of the present investigation, the analysis is a powerful tool in the case of comparison studies between homologous proteins. The substrate-binding loop region of CBR1 is highly conserved among orthologues of animal species in amino acid sequence levels. Although Sniffer, a Drosophila orthologue of CBR1, forms homo dimmer unlike monomer formed by other orthologues, the structure of the substrate-binding loop region of Sniffer (Protein Data Bank No.: 1SNY) was highly homologous to that of porcine testicular carbonyl reductase (PTCR; Protein Data Bank.: 1N5D; Ghosh et al. 2001), a porcine orthologue of CBR1 (Sgraja et al. 2004). Additionally, the structures of them show very high structural similarity to that of human orthologue (data not shown). On the other hand, although structural similarity between the animal orthologues of CBR3 cannot be analyzed due to little structural information and the formation of the loop structure of human CBR3 crystal (Protein Data Bank No.: 2HRB) in a crystal contact (Pilka et al. 2008; Miura et al. 2009a), there
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is high homology between the substrate-binding loop regions in amino acid sequence levels. Considering that carbonyl reductase activities of CBR1 and CBR3 are nearly conserved between animals, respectively (Nakajin et al. 1998; Ishikura et al. 1998; Miura et al. 2008, 2009a,b), the region may contribute to the common enzymatic differences between CBR1 and CBR3 in various animals. Many mutational studies on the catalysis of human CBR1 have been reported. For example, Wermuth et al. (Tinguely and Wermuth 1999) indicated that Cys227 of CBR1 was critical for catalysis, and the mutants (Cys227Ala, and Cys227Ser) exhibited higher Km values and lower kcat values toward substrates than the wild-type human CBR1. Baker et al. (Sciotti et al. 2000) showed that Thr241 in human CBR1 is important for autocatalytic modification with 2-oxoglutalate. In addition, rat ovarian carbonyl reductase exhibited lower Km values toward substrates than rat testicular carbonyl reductase due to Thr235 and Glu238 in the ovarian enzyme (Sciotti et al. 2006). Finally, it has been suggested that Val244 of human CBR3 plays an important role in binding with a cofactor, NADP(H) (Lakhman et al. 2005). These amino acid residues are in either the substrate-binding loop region or its surrounding region. Therefore, the region appeared to be significant for catalysis and coenzyme binding. However, we found that the substitution of the amino acid residue 230 alone of human CBR1 for the corresponding residue of human CBR3 has no apparent impact on carbonyl reductase activities of CBR3 (Miura et al. 2009a). Very recent study (El-Hawari et al. 2009) exhibited that, based on the strategy separated from our present study, mutation of human CBR3 to residues 230 and 236–244 of human CBR1 synergically induces high catalytic efficiencies for carbonyl reductase activities toward both isatin and 9, 10-phenanthrene-quinone. Results of a series of our investigations (the present study; Miura et al. 2009a) regarding carbonyl reductase activities of mutated enzymes are supported by their study. Our present region-swapping study clearly showed that unique properties of CBRs, with regard to not only carbonyl reductase activities but also coenzyme binding, are dominantly dependent on the properties of LirC (the substrate-binding regions) in CBR, and expanded the understanding of the role of the substrate-binding loop regions of CBR. Despite the complete conservation of a catalytic triad (S-YxxxK; Ser139, Tyr193, and Lys197) in both CBR1 and CBR3, CBR3 exhibited lower catalytic efficiencies of carbonyl reductase activity than CBR1 (Miura et al. 2008). However, the chimeric CBR3 containing LirC of CBR1 (CBR3LirC1) showed increased catalytic activity, suggesting that the catalytic triad of CBR3 is functionally enough to transfer the proton to substrates. Therefore, we cannot exclude the possibility that suitable substrates of CBR3 for its carbonyl reductase activity will be discovered in the future. Furthermore, since CBR1LirN3 showed CBR1like kinetics, LirN appeared not to contribute to the low carbonyl reductase activity of CBR3. This result may imply that the protonation states of catalytic residues were not influenced by the region, although LirN is located near the catalytic triad. LirC is also related to NADPH binding as indicated by the present fluorescence titration studies. Chimeric enzymes having LirC of CBR1 (CBR1, CBR3/1, and CBR3LirC) exhibited around 1 µM or less of Kd values. On the other hand, the Kd values of other enzymes, which include LirC of CBR3 (CBR3, CBR1/3, CBR1LirC3, and CBR1LirNC3) were clearly higher, although the values were diverse. It is noteworthy that the exchange of LirC of CBR3 alone converted the enzyme to exhibit a CBR3-like Kd value, and the tendency was also observed in the case of the exchange of LirC of CBR1. The crystal structure of human CBR1 (Protein Data Bank No.: 1WMA; Tanaka et al. 2005) and PTCR (Protein Data Bank No.: 1N5D; Ghosh et al. 2001) revealed that the amino acid residues in the substrate-binding loop region interact with the coenzyme directly or indirectly. The observations supported our present results regarding coenzyme binding. However, CBR1LirN3 also showed CBR3-like Kd values. The reason was not clear, but one of the possibilities may be that the substitution of LirN induces the
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structural alteration of a substrate-NADPH binding cleft in a micro domain and therefore indirectly influences a coenzyme-binding property, since the LirN is located near the cleft. Considering both the CBR1-like Kd value of CBR3LirC1 and the small difference between the Kd values of CBR1LirNC3 and CBR1LirC3, LirC was suggested to contribute to the specific property of NADPH binding in CBR. 3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (hydroxyl-PP) serves as an inhibitor for CBR1 most likely through the competitive inhibition of the substrate-binding loop region of CBR1 (Tanaka et al. 2005; Carlquist et al. 2008). Moreover, the derivative compound, 3-(7-isopropyl-4-(methylamino)-7H-pyrrolo [2,3-d]pyrimidin-5yl)phenol (hydroxyl-PP-Me) increased the sensitivity to the cell-killing activity of daunorubicin in a human lung carcinoma cell line, A549 cells (Tanaka et al. 2005). The metabolite of anthracycline (13-hydroxy anthracycline) inactivated by CBR1 was one of the causes of congestive heart failure induced by anthracycline treatment (Olson et al. 2003; Forrest et al. 2000). Despite the lowsequence identity (b30%) between members of SDR superfamily, CBR1 and CBR3 share particularly high identity (72%) in humans. Therefore, it seemed difficult to develop the specific inhibitors for CBR1. However, it is revealed by the present study that CBR1 is clearly separated from CBR3 in properties of the substrate-binding loop region. Thus, the region in CBR1 is a possible target of selective inhibitors that support anthracycline anti-cancer therapy through both increasing sensitivity to the drug and decreasing the toxic metabolites. Further kinetic analyses of our chimeric enzymes will provide insight into the development of such inhibitors. Conclusion Specific properties of human CBR1 and CBR3, regarding not only carbonyl reductase activities but also NADPH binding, are dependent on mainly the substrate-binding loop regions. Acknowledgements This work was supported in part by a research grant from Japan Heart Foundation Young Investigator's Research Grant (T.M.), and by a grant-in-aid for scientific research (C) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (T.M.). References Ax W, Soldan M, Koch L, Maser E. Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction. Biochemical Pharmacology 59 (3), 293–300, 2000. Blanco JG, Leisenring WM, Gonzalez-Covarrubias VM, Kawashima TI, Davies SM, Relling MV, Robison LL, Sklar CA, Stovall M, Bhatia S. Genetic polymorphisms in the carbonyl reductase 3 gene CBR3 and the NAD(P)H:quinine oxidoreductase 1 gene NQO1 in patients who developed anthracycline-related congestive heart failure after childhood cancer. Cancer 112 (12), 2789–2795, 2008. Bohren KM, von Wartburg JP, Wermuth B. Kinetics of carbonyl reductase from human brain. Biochemical Journal 244 (1), 165–171, 1987. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254, 1976. Carlquist M, Frejd T, Gorwa-Grauslund MF. Flavonoids as inhibitors of human carbonyl reductase 1. Chemico-Biological Interactions 174 (2), 98–108, 2008. El-Hawari Y, Favia AD, Pilka ES, Kisiela M, Oppermann U, Martin HJ, Maser E. Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by sitedirected mutagenesis. Chemico-Biological Interactions 178 (1–3), 234–241, 2009. Fan L, Goh BC, Wong CI, Sukri N, Lim SE, Tan SH, Guo JY, Lim R, Yap HL, Khoo YM, Iau P, Lee HS, Lee SC. Genotype of human carbonyl reductase CBR3 correlates with doxorubicin disposition and toxicity. Pharmacogenetics and Genomics 18 (7), 621–631, 2008. Forrest GL, Gonzalez B. Carbonyl reductase. Chemico-Biological Interactions 129 (1–2), 21–40, 2000. Forrest GL, Akman S, Doroshow J, Rivera H, Kaplan WD. Genomic sequence and expression of a cloned human carbonyl reductase gene with daunorubicin reductase activity. Molecular Pharmacology 40 (4), 502–507, 1991. Forrest GL, Gonzalez B, Tseng W, Li XL, Mann J. Human carbonyl reductase overexpression in the heart advances the development of doxorubicin-induced cardiotoxicity in transgenic mice. Cancer Research 60 (18), 5158–5164, 2000.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding☆ Takeshi Miura a,b,⁎, Toru Nishinaka a, Tomoyuki Terada a a b
Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan Department of Clinical Research, National Hospital Organization Hakodate Hospital, 18-16 Kawahara-cho, Hakodate, Hokkaido 041-8512, Japan
a r t i c l e
i n f o
Article history: Received 13 November 2008 Accepted 15 June 2009 Keywords: Short-chain dehydrogenase/reductase Anthracycline Chimera CBR Harr plot Swapping
a b s t r a c t Aims: Monomeric carbonyl reductase 1 (CBR1) and 3 (CBR3) are members of the short-chain dehydrogenase/ reductase superfamily, and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR3 exhibits a higher Km value toward NADPH and more limited carbonyl reductase activities than CBR1, although they are highly homologous to each other in amino acid sequence levels. In the present study, we investigated the origin of the different properties of the enzymes by analyses using several chimeric enzymes. Main Methods: Harr-plot analysis of the amino acid sequences was conducted and as a result, two low-identity regions between human CBR1 and CBR3 were found: these were designated as the N-terminal low-identity region (LirN) and the C-terminal low-identity region (LirC; the substrate-binding region). We genetically constructed chimeric enzymes while focusing on these regions. Key findings: Chimeric CBR1 possessing LirN of CBR3 (CBR1LirN3) exhibited CBR1-like activities but a low coenzyme affinity probably due to a structural alteration in a micro domain, whereas chimeric CBR1 including LirC of CBR3 (CBR1LirC3) was enzymatically similar to CBR3. Furthermore, CBR3LirC1 was similar to CBR1 in both enzymatic activities and coenzyme binding. Significance: These results suggested that LirC, i.e., the substrate-binding loop region, is the origin of the difference between human CBR1 and CBR3 in both catalytic and coenzyme-binding properties. © 2009 Elsevier Inc. All rights reserved.
Introduction The short-chain dehydrogenase/reductase (SDR) superfamily is a large group of enzymes with NAD(P)(H)-dependent oxidoreductase activities including hydroxysteroid dehydrogenase, prostaglandin (PG) oxidoreductase, retinal reductase/dehydrogenase, and carbonyl reductase (Hoffmann and Maser 2007; Oppermann 2007; Matsunaga et al. 2006; Forrest and Gonzalez 2000). In general, a low-sequence identity (b30%) is observed among the SDR enzymes, and the homologous region is restricted to the residues that are involved in the binding of coenzymes (the Rossmann-fold consensus sequence) and catalysis (Hoffmann and Maser 2007). Monomeric carbonyl reductases (CBRs) are members of the SDR superfamily. Two CBRs are found in humans: carbonyl reductase 1 (CBR1) (Wermuth et al. 1988) and carbonyl reductase 3 (CBR3) (Watanabe et al. 1998). The enzymes share high ☆ Part of this study was presented at the 8th International ISSX (the International Society for the Study of Xenobiotics) meeting on 9–12, Oct. 2007 (Sendai, Japan). Dr. E. Maser and his colleagues presented, on Jul. 8–12 2008, at the 14th International Meeting on Enzymology and Molecular Biology of Carbonyl Metabolism (Kranjska gora, Slovenia) regarding the origin of the difference between CBR1 and CBR3, and reached at essentially the same conclusion that we did. ⁎ Corresponding author. 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan. Tel.: +81 721 24 9983; fax: +81 721 24 9890. E-mail address: [email protected] (T. Miura). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.06.005
identity (72%) in their amino acid sequences. Both the N-terminal Rossmann-fold consensus sequence (GxxxGxG; 12–18 at amino acid residue) and the catalytic triad (S-YxxxK; Ser139, Tyr193, and Lys197) in the center and C-terminus of the enzymes are completely conserved. CBR1 is a reductase for endogenous and xenobiotic compounds using NADPH as a coenzyme, and has been known as a PG 9-keto reductase which is one of the physiological regulators of PG metabolism (Wermuth 1981), and also as a metabolizing enzyme for anthracycline anti-cancer drugs such as daunorubicin and doxorubicin (Kassner et al. 2008; Gavelová et al. 2008; Ax et al. 2000; Forrest et al. 1991). The metabolites of anthracycline (13-hydroxy anthracycline) that are inactivated by CBR1 are believed to induce congestive heart failure, which is sometimes lethal (Olson et al. 2003; Forrest et al. 2000). Furthermore, increased expression of CBR1 has caused the resistance to the drugs (Plebuch et al. 2007; Gonzalez et al. 1995). Therefore, the development of selective inhibitors to CBR1 is advisable for more efficient anthracycline dosage (Carlquist et al. 2008). On the other hand, the physiological roles of CBR3 remain unknown. Although genetic variants of CBR3 may correlate the pharmacokinetics of doxorubicin (Fan et al. 2008; Blanco et al. 2008), CBR3 is not an efficient reductase that reduces doxorubicin to 13-hydroxy doxorubicin (Kassner et al. 2008). In our recent investigations (Miura et al. 2008, 2009a,b), human CBR3 and the orthologues of Chinese hamsters and rats exhibited limited carbonyl reductase activities for the test substrates. Furthermore,
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the Km value of CBR3 toward NADPH was higher than that of CBR1. These observations suggested that CBR1 and CBR3 play different physiological roles although they are highly homologous. The clarification of the origin of the enzymatic differences between CBR1 and CBR3 is useful not only in terms of fostering a deeper understanding of both the enzymatic mechanism and physiological roles of the enzymes but also for the development of CBR1-selective inhibitors. Thus, we bioinformatically analyzed their amino acid sequences and identified two low-identity regions. In the present study, the contribution of these low-identity regions to the enzymatic differences between the enzymes was investigated by conducting analyses of several chimeric enzymes that may become powerful tools for the development of selective inhibitors. Materials and methods Materials All reagents in this study were of the highest grade that is commercially available. Construction of bacterial-expression vectors for chimeric CBR Bacterial-expression vectors for CBR1 and CBR3 (pET-hCBR1 and pET-hCBR3, respectively) were previously described (Miura et al. 2008). Bacterial-expression vectors for chimeric proteins were designated as indicated in Fig. 2. The N-terminal region of CBR3 cDNA was amplified by polymerase chain reaction (PCR) using hmhCBR1f03(NdeI) (5′-cgc ata tgt tcc gcg cgc cc-3′; bold, NdeI-recognition site) and mthCBR3r03 (5′-tcg ggt acc aaa aaa att tgt ctt cag tgt ca-3′; bold, KpnI-recognition sequence) as primers, and excised with NdeI and KpnI. The DNA fragment was inserted into the NdeI–KpnI site in pET-hCBR1. The resulting vector was termed pET-hCBR3/1. Similarly, for the construction of pEThCBR1/3, the C-terminal region of CBR3 cDNA was amplified by PCR using mthCBR3f03 (5′-ttt ggt acc aga aac atg tgc aac gag tta-3′; bold, the KpnI-recognition sequence) and hmhCBR3r03(EcoRI) (5′-acg aat tca gga caa ggt aca aaa tgg ggc-3′; bold, the EcoRI-recognition site) as primers, excised with KpnI and EcoRI, and inserted into the KpnI– EcoRI site in pET-hCBR1. The constructions of pET-hCBR1LirN3, pET-hCBR1LirC3, and pEThCBR3LirC1 were performed with the In-fusion PCR cloning kit (Clontech Laboratories, Inc., Palo Alto, CA, USA): DNA fragments of the chimeric region (LirN or LirC) in hCBR1 or hCBR3 were reacted with the products of inverted tail-to-tail PCR using pET-hCBR1 or pET-hCBR3 as templates. The primers were as follows; mthCBR1f12 (5′-agt gag acc atc act gag gag gag ctg-3′), mthCBR1r13 (5′-aga tac gtt cac cac tct ccc ttg ggg3′), mthCBR3f07 (5′-gtg gtg aac gta tct agt ttg cag tgt tt agg-3′), and mthCBR3r07 (5′-agt gat ggt ctc act gtg gaa cct ttc ctg-3′) for pEThCBR1LirN3; mthCBR1f05 (5′-acc ctg tgt act tgg ccc tt-3′), mthCBR1r06 (5′-gca gca agg cat tca gga gga tc-3′), mthCBR3f05 (5′-ctg aat gcc tgc tgc cca gga cca gtg ccg-3′), and mthCBR3r05 (5′-caa gta cac agg ggt ctc agc ccc ctg ctc-3′) for pET-hCBR1LirC3; mthCBR3f04 (5′-acc cct gtc tac ttg gcc ctc-3′), mthCBR3r04 (5′-gca gca cgc att cac cag aat c-3′), mthCBR1f11 (5′-gtg aat gcg tgc tgc cca ggg tgg gtg aga-3′), and mthCR1r12 (5′-caa gta gac agg ggt ctc tgc acc ttc ttc-3′) for pET-hCBR3LirC1. pET-hCBR1LirNC3 was constructed by the ligation of the NdeI/KpnI fragment of pET-hCBR1LirN3 to the NdeI/KpnI site of pEThCBR1LirC3. Bacterial expression and purification of recombinant wild-type and chimeric enzymes
When the absorbance of the culture at 600 nm was 0.5–0.8, isopropyl-βD-thiogalactopyranoside (IPTG) was added to the medium at a concentration of 0.2 mM, and the cells were cultured for an additional 3–4 h. The bacterial cells were then harvested, washed with 20 mM sodiumphosphate buffer, pH 7.5 (buffer A), resuspended with 30 ml of buffer A, and then sonicated. After centrifugation of the crude extract at 17,000 ×g for 30 min, the supernatant was subjected to purification using Ni++ chelating affinity column chromatography (bed volume, 1 ml). After equilibrating the column with wash buffer 1 (50 mM potassium-phosphate buffer, pH 8.0, with 300 mM KCl and 5 mM imidazole), the supernatant was loaded. The column was washed with 6 ml of wash buffer 1 and then 6 ml of wash buffer 2 (50 mM potassium-phosphate buffer, pH 8.0, with 300 mM KCl and 10 mM imidazole). The protein adsorbed to the affinity resin was eluted with elution buffer (50 mM potassium-phosphate buffer, pH 8.0, with 300 mM KCl and 250 mM imidazole). The eluted protein was gelfiltrated with Bio-Gel P-6 (Bio-Rad Laboratories, Hercules, CA, USA) preequilibrated with buffer A, and then dithiothreitol (DTT) was added at a final concentration of 5 mM. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard (Bradford 1976). The purified CBR proteins exhibited a single band on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue G-250 (CBB) staining. Determination of enzymatic activity The reaction mixture for the reductase activity consisted of 100 mM sodium-phosphate buffer, pH 6.5,130 µM NADPH, and the substrate. The reaction was initiated by the addition of an appropriate amount of enzyme. The enzymatic activity was monitored by measuring the decrease in absorbance at 340 nm depending on the conversion of NADP (H) at 25 °C. Five concentrations of each substrate were assayed at the minimum, and each concentration was measured at least three times. These reactions were performed at a total volume of 1 ml. Menadione and 4-benzoylpyridine stock solutions were prepared in methanol. The final concentration of methanol in the reaction was not more than 10% (v/v). The apparent Km and kcat values were determined by fitting the initial velocities to the Michaelis–Menten equation with SigmaPlot (Systat Software, Inc., Richmond, CA, USA) based on a molar extinction coefficient for NADPH of 6.22 × 103 M− 1 cm− 1. Each kinetic constant was the average of three measurements. Coenzyme binding To determine coenzyme dissociation constants, we measured the changes in the intrinsic enzyme fluorescence on the incremental addition of coenzymes on a spectrofluorophotometer (RF-5300PC; Shimadzu Co. Ltd., Kyoto, Japan). To ensure that the volume of coenzyme added did not exceed 3% of the total volume, three stock solutions of the coenzyme were prepared: 13 mM, 6.5 mM, and 1.3 mM. A 4 × 10-mm cuvette was used, and each 3 ml sample contained 60 µg of proteins in 100 mM sodium-phosphate buffer, pH 6.5 at 25 °C. The samples were excited at 290 nm and the fluorescence emission was scanned at 335 nm. Hill's equation was fitted to the fluorescence decreases that were dependent on coenzyme binding in order to estimate the corresponding dissociation constants for the wild-type and chimeric enzymes using SigmaPlot. Kd values were obtained from three experiments performed with at least two independent protein preparations. Results Identification of low-identity regions between CBR1 and CBR3
The bacterial cells [E. coli, BL21(DE3)pLysE] transformed by various bacterial-expression vectors were precultured overnight in Luria (L)-broth containing 25 µg/ml kanamycin at 37 °C with vigorous shaking, and then further cultured in 25 volumes of L-broth under the same conditions.
Regarding the orthologues of humans, Chinese hamsters, and rats, CBR3 exhibits extremely lower catalytic carbonyl reductase activity than CBR1, although both enzymes share high identity (70–75%) in
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Fig. 2. Structures of chimeric enzymes. The amino acid sequences of CBR1 and CBR3 were shown as opened and closed boxes, respectively.
both LirN and LirC) includes the enzymatic determinant of specific properties of CBRs. Specific properties of CBRs were dependent on the substrate-binding loop region
Fig. 1. (A) Harr-plot analysis for human CBR1 and CBR3 in their amino acid sequences. Harr-plot analysis was performed under the following conditions: unit size to compare, 5; dot plot score, 3.00. Two low-identity regions were termed the N-terminal lowidentity region (LirN; 140–159 at amino acid residue) and the C-terminal low-identity region (LirC; 230–244 at amino acid residue), respectively. (B) Sequence alignment of LirN between CBR1 and CBR3. (C) Sequence alignment of LirC between CBR1 and CBR3.
their amino acid sequences. To investigate the origin of the enzymatic difference, Harr-plot analysis (Harr et al. 1982; Staden 1982) was used to compare the amino acid sequences (Fig. 1; performed by GENETYX software; Genetyx Co. Ltd., Tokyo, Japan). The analysis is a twodimensional graphical homology search in nucleotide and amino acid sequences. Harr-plot analysis between CBR1 and CBR3 revealed a discontinuous-line pattern, and two low-identity regions were found as opening spaces in the line (Fig. 1A). One was the region near catalytic Ser-139 (140–159 at amino acid residue; the N-terminal lowidentity region, LirN), and formed two α helix structures, which are αEF1 and αEF2 as considered from the crystal structures of CBR1 (Protein Data Bank No.: 1WMA) and CBR3 (Protein Data Bank No.: 2HRB) (Fig. 1B). The other was the substrate-binding loop region (230–244 at amino acid residue) (Fig. 1C), which was designated as the C-terminal low-identity region (LirC). The identification of these two low-identity regions in CBR prompted us to study the involvement of these regions in the enzymatic properties by characterizing the chimeric enzymes between CBR1 and CBR3. Contribution of the C-terminal region of CBRs for enzymatic properties First, we examined whether the two regions in CBR1 and CBR3 can affect their carbonyl reductase activities. Chimeric enzymes, CBR3/1 and CBR1/3, contain both LirN and LirC of CBR1 and CBR3, respectively (Fig. 2). The kinetic constants of these enzymes toward test substrates (menadione and 4-benzoylpyridine) for carbonyl reductase activity were determined (Table 1). CBR3/1 and CBR1/3 exhibited kinetic constants similar to CBR1 and CBR3, respectively, suggesting that the C-terminal region of CBRs (118–277 at amino acid residue; containing
The above-mentioned results implied the possibility that either LirN or LirC affects the specific properties of CBR1 and CBR3. Therefore, we characterized the chimeric enzymes more precisely (Table 1). The chimeric enzyme of CBR1 containing LirN of CBR3 (CBR1LirN3) exhibited almost CBR1-like kinetic constants, although its catalytic efficiencies were low as compared to those of CBR1. On the other hand, CBR1LirC3 and CBR1LirNC3 showed low catalytic efficiencies, which were similar to CBR3. Moreover, the exchange of LirC in CBR3 for that of CBR1 (CBR3LirC1) increased the kcat and catalytic efficiencies of the enzymes. Similarly, the Km values toward NADPH were dependent on LirC, not LirN. These results suggested that specific properties for catalysis of CBR1 and CBR3 are dependent on LirC, i.e., the substratebinding loop region. Coenzyme binding properties of wild-type and chimeric enzymes The Km values of CBR3 toward NADPH were reported to be higher values than those of CBR1 (an order of 1 µM) (Miura et al. 2008; Lakhman et al. 2005; Usami et al. 2001; Bohren et al. 1987), and we obtained reproducible results (Table 1). We used chimeric enzymes to determine the critical region(s) where such a coenzyme-binding difference between CBR1 and CBR3 exists. The binding of the enzymes to NADPH was measured by the decrease in the intrinsic enzyme fluorescence on the incremental addition of NADPH. The fluorescence spectra of CBR1 and CBR3 without NADPH are shown in Fig. 3A. Table 1 Menadione CBR1 CBR3/1 CBR1LirN3 CBR1LirC3 CBR1LirNC3 CBR3LirC1 CBR1/3 CBR3
4-benzoylpyridine
NADPH
Km
kcat
kcat/Km
Km
kcat
kcat/Km
Km
Kd
30 49 34 49 42 45 65 54
410 110 41 0.079 0.053 59 0.88 0.32
14 2.2 1.2 0.0016 0.0013 1.3 0.014 0.0059
360 400 380 470 420 450 360 300
400 270 140 0.37 0.20 64 1.2 0.70
1.1 0.68 0.37 0.00079 0.00048 0.14 0.0033 0.0023
6.2 5.3 7.4 16 19 5.8 32 26
1.2 0.21 5.7 7.1 7.3 0.66 21 7.5
Km and Kd values were shown as µM. kcat values were shown as min− 1. To investigate kinetic constants toward coenzymes, the concentration of menadione was held at 200 µM. Standard deviation of each value was within 25%.
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Kd values that were distinct from those of CBR1: the Kd values of these chimeric enzymes were higher than those of CBR1. Therefore, the Kd value of CBR toward NADPH was suggested to be dependent on LirC. The Kd values of wild-type and chimeric enzymes toward NADPH, which are lower as compared to the Km values, might be due to insufficient saturation of the substrate, menadione. Discussion
Fig. 3. Fluorescence titrations of CBR for NADPH. (A) Fluorescence spectra of CBR1 and CBR3 with the excitation at 290 nm are shown. The intensities of fluorescence at 335 nm at each protein were supposed to be 1.0 as a standard in order to observe the differences in the fluorescence peak. Solid line, CBR1; broken line, CBR3. (B, C) Typical fitting curves are indicated. The decreases in relative fluorescence (ex. 290 nm/em. 335 nm) of CBR1 (B) and CBR3 (C) fitted to the curves of Hill's equation. The Hill plot was in each graph to consider the degree of linear regression.
The wavelength at the fluorescence peak of CBR3 slightly differed from that of CBR1.With regard to the wavelength to observe the intrinsic fluorescence of CBR1 and CBR3, the Kd values estimated by the fluorescence decrease at 335 nm on the addition of NADPH were essentially the same as the values estimated by the fluorescence decrease at 325, 330, 340, and 345 nm (data not shown). Therefore, the data at 335 nm were used in the present study to measure the Kd values. The Kd value of CBR1 toward NADPH was in good agreement with a previous report (Bohren et al. 1987). The Kd value of CBR1 was clearly lower than that of CBR3. Moreover, CBR3/1 and CBR3LirC1 exhibited Kd values similar to CBR1. On the other hand, chimeric enzymes that have the LirC of CBR3 (CBR1/3, CBR1LirC3, and CBR1LirNC3) showed
CBRs are members of the SDR superfamily and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR1 and CBR3 share high identity (70–75%) in their amino acid sequences (Hoffmann and Maser 2007; Oppermann 2007; Matsunaga et al. 2006; Forrest and Gonzalez 2000). CBR1 has been well characterized from the views of both drug metabolism and enzymology. A recent study (Kassner et al. 2008) revealed that in humans, CBR1 is a dominant enzyme that metabolizes doxorubicin, an anticancer drug, in the livers whereas CBR3 does not contribute to the inactivation of the drug. Furthermore, in our investigations (Miura et al. 2008, 2009a,b; Terada et al. 2001, 2003), CBR3 exhibited lower carbonyl reductase activities toward test substrates than CBR1 in humans, Chinese hamsters, and rats. These results suggested that the limited carbonyl reductase activity of CBR3 is a common property among animal species. Moreover, although both the coenzymebinding motif (the Rossmann-fold consensus sequence, GxxxGxG in the N-terminus of the proteins) and the “NADPH-specificity motif KAR” (Hoffmann and Maser 2007) near the Rossmann-fold consensus sequence are highly conserved between CBR1 and CBR3, the Km value of CBR3 toward NADPH was suggested to be highly distinct from that of CBR1. In the present study, in order to clarify the origin of these differences, we bioinformatically searched low-identity regions between CBR1 and CBR3. Based on the result (Fig. 1), we genetically constructed and characterized several chimeric enzymes, and finally, identified the substrate-binding loop region in CBR1 and CBR3 as the region that is responsible for the specific properties of the two enzymes. Substrates binding with the SDR enzymes are expected to be followed by both the swinging and accommodating of the loop without an apparent conformational change of the loop structure (Tanaka et al. 1996), suggesting that unique properties of CBR1 and CBR3 are mainly contributed by the structural differences between the loop structures. Low-identity regions that were investigated in the present study were rendered by the result of Harr-plot analysis (Fig. 1). The analysis is a graphical homology search (Harr et al. 1982; Staden 1982). Since the result is graphically provided, the analysis simplifies the evaluation of the result from homology search; therefore, the analysis has the power to appeal the existence of local regions that exhibit high or low identity for the human eyes, which is usually a weak point of the simple homology search. Thus, as we easily identified low-identity regions as targets of the present investigation, the analysis is a powerful tool in the case of comparison studies between homologous proteins. The substrate-binding loop region of CBR1 is highly conserved among orthologues of animal species in amino acid sequence levels. Although Sniffer, a Drosophila orthologue of CBR1, forms homo dimmer unlike monomer formed by other orthologues, the structure of the substrate-binding loop region of Sniffer (Protein Data Bank No.: 1SNY) was highly homologous to that of porcine testicular carbonyl reductase (PTCR; Protein Data Bank.: 1N5D; Ghosh et al. 2001), a porcine orthologue of CBR1 (Sgraja et al. 2004). Additionally, the structures of them show very high structural similarity to that of human orthologue (data not shown). On the other hand, although structural similarity between the animal orthologues of CBR3 cannot be analyzed due to little structural information and the formation of the loop structure of human CBR3 crystal (Protein Data Bank No.: 2HRB) in a crystal contact (Pilka et al. 2008; Miura et al. 2009a), there
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is high homology between the substrate-binding loop regions in amino acid sequence levels. Considering that carbonyl reductase activities of CBR1 and CBR3 are nearly conserved between animals, respectively (Nakajin et al. 1998; Ishikura et al. 1998; Miura et al. 2008, 2009a,b), the region may contribute to the common enzymatic differences between CBR1 and CBR3 in various animals. Many mutational studies on the catalysis of human CBR1 have been reported. For example, Wermuth et al. (Tinguely and Wermuth 1999) indicated that Cys227 of CBR1 was critical for catalysis, and the mutants (Cys227Ala, and Cys227Ser) exhibited higher Km values and lower kcat values toward substrates than the wild-type human CBR1. Baker et al. (Sciotti et al. 2000) showed that Thr241 in human CBR1 is important for autocatalytic modification with 2-oxoglutalate. In addition, rat ovarian carbonyl reductase exhibited lower Km values toward substrates than rat testicular carbonyl reductase due to Thr235 and Glu238 in the ovarian enzyme (Sciotti et al. 2006). Finally, it has been suggested that Val244 of human CBR3 plays an important role in binding with a cofactor, NADP(H) (Lakhman et al. 2005). These amino acid residues are in either the substrate-binding loop region or its surrounding region. Therefore, the region appeared to be significant for catalysis and coenzyme binding. However, we found that the substitution of the amino acid residue 230 alone of human CBR1 for the corresponding residue of human CBR3 has no apparent impact on carbonyl reductase activities of CBR3 (Miura et al. 2009a). Very recent study (El-Hawari et al. 2009) exhibited that, based on the strategy separated from our present study, mutation of human CBR3 to residues 230 and 236–244 of human CBR1 synergically induces high catalytic efficiencies for carbonyl reductase activities toward both isatin and 9, 10-phenanthrene-quinone. Results of a series of our investigations (the present study; Miura et al. 2009a) regarding carbonyl reductase activities of mutated enzymes are supported by their study. Our present region-swapping study clearly showed that unique properties of CBRs, with regard to not only carbonyl reductase activities but also coenzyme binding, are dominantly dependent on the properties of LirC (the substrate-binding regions) in CBR, and expanded the understanding of the role of the substrate-binding loop regions of CBR. Despite the complete conservation of a catalytic triad (S-YxxxK; Ser139, Tyr193, and Lys197) in both CBR1 and CBR3, CBR3 exhibited lower catalytic efficiencies of carbonyl reductase activity than CBR1 (Miura et al. 2008). However, the chimeric CBR3 containing LirC of CBR1 (CBR3LirC1) showed increased catalytic activity, suggesting that the catalytic triad of CBR3 is functionally enough to transfer the proton to substrates. Therefore, we cannot exclude the possibility that suitable substrates of CBR3 for its carbonyl reductase activity will be discovered in the future. Furthermore, since CBR1LirN3 showed CBR1like kinetics, LirN appeared not to contribute to the low carbonyl reductase activity of CBR3. This result may imply that the protonation states of catalytic residues were not influenced by the region, although LirN is located near the catalytic triad. LirC is also related to NADPH binding as indicated by the present fluorescence titration studies. Chimeric enzymes having LirC of CBR1 (CBR1, CBR3/1, and CBR3LirC) exhibited around 1 µM or less of Kd values. On the other hand, the Kd values of other enzymes, which include LirC of CBR3 (CBR3, CBR1/3, CBR1LirC3, and CBR1LirNC3) were clearly higher, although the values were diverse. It is noteworthy that the exchange of LirC of CBR3 alone converted the enzyme to exhibit a CBR3-like Kd value, and the tendency was also observed in the case of the exchange of LirC of CBR1. The crystal structure of human CBR1 (Protein Data Bank No.: 1WMA; Tanaka et al. 2005) and PTCR (Protein Data Bank No.: 1N5D; Ghosh et al. 2001) revealed that the amino acid residues in the substrate-binding loop region interact with the coenzyme directly or indirectly. The observations supported our present results regarding coenzyme binding. However, CBR1LirN3 also showed CBR3-like Kd values. The reason was not clear, but one of the possibilities may be that the substitution of LirN induces the
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structural alteration of a substrate-NADPH binding cleft in a micro domain and therefore indirectly influences a coenzyme-binding property, since the LirN is located near the cleft. Considering both the CBR1-like Kd value of CBR3LirC1 and the small difference between the Kd values of CBR1LirNC3 and CBR1LirC3, LirC was suggested to contribute to the specific property of NADPH binding in CBR. 3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (hydroxyl-PP) serves as an inhibitor for CBR1 most likely through the competitive inhibition of the substrate-binding loop region of CBR1 (Tanaka et al. 2005; Carlquist et al. 2008). Moreover, the derivative compound, 3-(7-isopropyl-4-(methylamino)-7H-pyrrolo [2,3-d]pyrimidin-5yl)phenol (hydroxyl-PP-Me) increased the sensitivity to the cell-killing activity of daunorubicin in a human lung carcinoma cell line, A549 cells (Tanaka et al. 2005). The metabolite of anthracycline (13-hydroxy anthracycline) inactivated by CBR1 was one of the causes of congestive heart failure induced by anthracycline treatment (Olson et al. 2003; Forrest et al. 2000). Despite the lowsequence identity (b30%) between members of SDR superfamily, CBR1 and CBR3 share particularly high identity (72%) in humans. Therefore, it seemed difficult to develop the specific inhibitors for CBR1. However, it is revealed by the present study that CBR1 is clearly separated from CBR3 in properties of the substrate-binding loop region. Thus, the region in CBR1 is a possible target of selective inhibitors that support anthracycline anti-cancer therapy through both increasing sensitivity to the drug and decreasing the toxic metabolites. Further kinetic analyses of our chimeric enzymes will provide insight into the development of such inhibitors. Conclusion Specific properties of human CBR1 and CBR3, regarding not only carbonyl reductase activities but also NADPH binding, are dependent on mainly the substrate-binding loop regions. Acknowledgements This work was supported in part by a research grant from Japan Heart Foundation Young Investigator's Research Grant (T.M.), and by a grant-in-aid for scientific research (C) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (T.M.). References Ax W, Soldan M, Koch L, Maser E. Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction. Biochemical Pharmacology 59 (3), 293–300, 2000. Blanco JG, Leisenring WM, Gonzalez-Covarrubias VM, Kawashima TI, Davies SM, Relling MV, Robison LL, Sklar CA, Stovall M, Bhatia S. Genetic polymorphisms in the carbonyl reductase 3 gene CBR3 and the NAD(P)H:quinine oxidoreductase 1 gene NQO1 in patients who developed anthracycline-related congestive heart failure after childhood cancer. Cancer 112 (12), 2789–2795, 2008. Bohren KM, von Wartburg JP, Wermuth B. Kinetics of carbonyl reductase from human brain. Biochemical Journal 244 (1), 165–171, 1987. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254, 1976. Carlquist M, Frejd T, Gorwa-Grauslund MF. Flavonoids as inhibitors of human carbonyl reductase 1. Chemico-Biological Interactions 174 (2), 98–108, 2008. El-Hawari Y, Favia AD, Pilka ES, Kisiela M, Oppermann U, Martin HJ, Maser E. Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by sitedirected mutagenesis. Chemico-Biological Interactions 178 (1–3), 234–241, 2009. Fan L, Goh BC, Wong CI, Sukri N, Lim SE, Tan SH, Guo JY, Lim R, Yap HL, Khoo YM, Iau P, Lee HS, Lee SC. Genotype of human carbonyl reductase CBR3 correlates with doxorubicin disposition and toxicity. Pharmacogenetics and Genomics 18 (7), 621–631, 2008. Forrest GL, Gonzalez B. Carbonyl reductase. Chemico-Biological Interactions 129 (1–2), 21–40, 2000. Forrest GL, Akman S, Doroshow J, Rivera H, Kaplan WD. Genomic sequence and expression of a cloned human carbonyl reductase gene with daunorubicin reductase activity. Molecular Pharmacology 40 (4), 502–507, 1991. Forrest GL, Gonzalez B, Tseng W, Li XL, Mann J. Human carbonyl reductase overexpression in the heart advances the development of doxorubicin-induced cardiotoxicity in transgenic mice. Cancer Research 60 (18), 5158–5164, 2000.
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