Investigation of the role of the amino acid residue at position 230 for catalysis in monomeric carbonyl reductase 3

Chemico-Biological Interactions 178 (2009) 211–214

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Investigation of the role of the amino acid residue at position 230 for catalysis in monomeric carbonyl reductase 3 Takeshi Miura a,c,∗ , Yuma Itoh a , Masahito Takada a , Hidenobu Tsutsui b , Tokihito Yukimura b , Toru Nishinaka a , Tomoyuki Terada a a

Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan Laboratory of Clinical Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan Department of Clinical Research, National Hospital Organization Hakodate Hospital, 18-16 Kawahara-cho, Hakodate, Hokkaido 041-8512, Japan b c

a r t i c l e

i n f o

Article history: Received 1 August 2008 Received in revised form 26 September 2008 Accepted 1 October 2008 Available online 15 October 2008 Keywords: Carbonyl reductase CBR1 CBR3 Short-chain dehydrogenase/reductase SDR

a b s t r a c t Monomeric carbonyl reductase 3 (CBR3) is a member of the short-chain dehydrogenase/reductase family. CBR3 exhibits much lower activity than monomeric carbonyl reductase 1 (CBR1) in humans and Chinese hamsters although they are highly homologous to each other in amino acid sequence levels. In the present study, we first cloned the CBR3 gene of rat origin (rCBR3), and characterized its enzymatic activity. rCBR3 also exhibited a limited catalytic efficiency similarly to the other CBR3 orthologues of humans and Chinese hamsters. Among the CBR3 orthologues, the human enzyme showed considerably lower activity. Compared with the amino acid sequences of CBR1 and CBR3 among humans, rats, Chinese hamsters, and mice, the tryptophan residue at position 230 is highly conserved while human CBR3 possesses rigid amino acid, proline, at that position instead. Thus, the Trp-230 was expected to be one of the important residues for catalysis since it locates in the hinge region at the substrate-binding loop. The substitution of tryptophan for proline in hCBR3 failed to affect the enzymatic characteristics. Similarly, the substitution of proline for tryptophan in either Chinese hamster CBR3 (CHCR3) or rCBR3 showed no significant change in the catalytic properties. These results suggest that limited catalytic efficiency of carbonyl reductase activity of CBR3 is a common property among animal species, and the substitution of the amino acid residue at position 230 alone has no apparent impact on their enzymatic activities. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Monomeric carbonyl reductase 3 (CBR3) [1] in the shortchain dehydrogenase/reductase (SDR) family is highly homologous to monomeric carbonyl reductase 1 (CBR1) [2] in its amino acid sequence levels [3–6]. In humans and Chinese hamsters, despite high homology between CBR3 and CBR1, CBR3 (human CBR3, hCBR3; Chinese hamster CBR3, CHCR3) exhibited a much lower catalytic efficiency of carbonyl reductase activity than CBR1 (human CBR1, hCBR1; Chinese hamster CBR1, CHCR1; Chinese hamster CBR2 [an orthologue of CBR1, not CBR2], CHCR2) [6–8]. CBR1 has been recognized as a metabolizing enzyme for anthracycline anti-cancer drugs such as daunorubicin, and doxorubicin

∗ Corresponding author at: Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan. Tel.: +81 721 249983; fax: +81 721 249890. E-mail address: [email protected] (T. Miura). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.10.005

[9–12], and the metabolites are believed to induce the congestive heart failure as a side effect [13,14]. Based on the high homology between hCBR1 and hCBR3, hCBR3 was also presumed to metabolize anthracycline [15]. However, although genetic polymorphisms of hCBR3 might correlate with anthracycline-related congestive heart failure [15,16], the results by Blanco et al. [16] revealed low catalytic activity of hCBR3 toward doxorubicin. Finally, Kassner et al. [17] revealed no contribution of hCBR3 for doxorubicin metabolism. CBR3 may be involved in the metabolism of steroids [6–8]. In the present study, we cloned and characterized rat CBR3 (rCBR3) in order to investigate the enzymatic mechanism of hCBR3 by comparing the enzymatic properties of some orthologues of the protein, and revealed the low activity of hCBR3 among its orthologues. Compared with the amino acid sequences of CBR1 and CBR3 among humans, rats, Chinese hamsters, and mice, the tryptophan residue at position 230 is highly conserved while human CBR3 possesses rigid amino acid, proline, at that position instead. Therefore, the Trp-230 was expected to be one of the important residues for catalysis since it locates in the hinge region at the substrate-binding

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loop. Thus, we carried out the site-directed mutagenesis study for the residue. 2. Materials and methods

albumin as a standard [19]. 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.

2.1. Materials

2.4. Determination of enzymatic activity

All reagents in this study were of the highest grade commercially available.

The reaction mixture for the reductase activity consisted of 100 mM sodium-phosphate buffer, pH 6.5, 130 ␮M 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. These reactions were carried out at a total volume of 1 ml. The apparent Km and kcat values were determined by fitting the initial velocities to the Michaelis–Menten equation with SigmaPlot (Systat Software, Inc., Richimond, CA, USA).

2.2. Construction of bacterial expression vectors for CBR3 In order to amplify the cDNA of the rCBR3 gene, polymerase chain reaction (PCR) was performed using a rat liver cDNA pool (Clontech Laboratories, Inc., CA, USA) from Sprague-Dawley (SD) rats as a template, using the following primers: mtrCBR3f01, 5 -gcc gcg gcc gcc acc aag tca gga tag aag g-3 , and mtrCBR3r01, 5 -gcc gga tcc tgg tcc gtg tgt ccc tct ga-3 . The products were excised with BamHI and NotI, and inserted into the pET-28a bacterial expression vector (pET-rCBR3). Other bacterial expression vectors for hCBR3 (pET-hCBR3) and CHCR3 (pET-CHCR3) were previously described [6–8]. These vectors express each CBR3 protein with a His cluster at the amino terminus in the protein. Site-directed mutagenesis (P230W for hCBR3, and W230P for rCBR3 and CHCR3) was carried out for pET-hCBR3, pET-CHCR3, and pET-rCBR3 by the method of Higuchi et al. [18]. The primers were listed as follows: hmhCBR1f03(NdeI) (5 -cgc ata tgt tcc gcg cgc cc3 ), hmhCBR3r02(EcoRI) (5 -tag aat tca gca agc tcc gaa gca gac-3 ), mthCBR3f12 (5 -tgc cca gga tgg gtg aag aca-3 ), and mthCBR3r10 (5 -tgt ctt cac cca tcc tgg gca-3 ) for the mutation of hCBR3; YSCRf04 (5 -cgt gtg gat ccg ata tgt cgt cct g-3 ), YSCRr05 (5 -cat ttg gat ccc tct cga gca gtt tac-3 ), mtCHCRf01 (5 -gcc cag ggc cgg tga aga ccg-3 ), and mtCHCRr01 (5 -cgg tct tca ccg gcc ctg ggc-3 ) for the mutation of CHCR3; and mtrCBR3f01, mtrCBR3r01, mtCHCRf01, and mtCHCRr01 for the mutation of rCBR3. The resulting plasmids were named pET-hCBR3 P230W, pET-CHCR3 W230P, and pET-rCBR3 W230P, respectively. 2.3. Bacterial expression and purification of recombinant CBR3 The pET-CBR3-transformed bacterial cells [E. coli, BL21(DE3) pLysE] were precultured overnight in Luria (L)-broth containing 25 ␮g/ml kanamycin at 37 ◦ C with vigorous shaking, and then further cultured in 25 volumes of L-broth under the same conditions. When the absorbance of the culture at 600 nm was 0.5–0.8, isopropyl-␤-d-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 sodium-phosphate 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 Ni2+ 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 gel-filtrated 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

3. Results and discussion 3.1. The amino acid sequence of rCBR3 The cDNA of rCBR3 was cloned from a liver cDNA library using primers designed based on the rat genome nucleotide sequence. The amino acid sequence deduced from isolated cDNA of rCBR3 was identical to the sequence in GenBank accession no. NP 001100580.1. rCbr3 encodes the protein consisting of 277 amino acids. The gene shared high identity with other orthologues in both nucleotide (cDNA) and deduced amino acid sequences levels (Table 1). 3.2. Enzymatic characteristics of rCBR3 Kinetic characteristics of rCBR3 toward menadione and 4benzoylpyridine (4-BP) were investigated. The Km value of rCBR3 toward menadione was too high to calculate kinetic constants. Both the Km value and the catalytic efficiency of rCBR3 toward 4-BP were also higher than those of hCBR3 (Table 2). The enzyme exhibited 3-keto steroid reductase activity, although the activities were not sufficient to calculate kinetic data with the spectrophotometric analyses (Table 3). 3.3. Effect of the substitution of the amino acid residue at position 230 Both catalytic efficiencies and kcat values of hCBR3 toward the substrates were lower than those of the other orthologues (Table 2; kcat , approximately 10 folds; catalytic efficiency, several folds). Therefore, we investigated the mechanism underlying the lower catalytic activity of hCBR3 while focusing on the substrate-binding loop region. The substrate-binding loop structure in the SDR family may play a critical role in the catalysis of the enzyme [20]. The structure between ␤F and ␣G in hCBR3 was shorter than that in hCBR1, Table 1 Percent identity among CBR3 orthologues of human, Chinese hamster, rat, and mouse in the nucleotide and amino acid sequences level. a.a.

n.a. Rat

Rat Human Chinese hamster Mouse

84.8 94. 2 97.4

Human

Chinese hamster

Mouse

74.5

87.1 76.5

92.4 76.6 86.7

86.6 85.2

93.9

n.a., nucleic acid sequence levels; a.a., amino acid sequence levels.

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Table 2 Kinetic constants of CBR3s and their mutants. Menadione

4-Benzoylpyridine

Menadione

4-Benzoylpyridine

rCBR3 Km kcat kcat /Km

>300 – 0.030a

1500 14 0.0093

rCBR3 W230P Km kcat kcat /Km

>300 – 0.032a

1800 11 0.0061

hCBR3 Km kcat kcat /Km

67 0.66 0.0099

530 0.62 0.0012

hCBR3 P230W Km kcat kcat /Km

79 0.73 0.0092

580 0.55 0.00095

CHCR3 Km kcat kcat /Km

89 2.9 0.033

620 3.8 0.0061

CHCR3 W230P Km kcat kcat /Km

97 2.5 0.026

540 3.9 0.0072

Km and kcat values are shown in pm and mm1, respectively. Each value represents the mean obtained from three experiments. S.D. of experiments was within 25%. a Catalytic efficiencies were provided from the slope of the initial velocity versus substrate concentration curve.

Table 3 Specificity toward steroids of CBR3s and their mutants.

Menadione 5␣-androstane-3,17-dione 5␣-androstane-3␣-ol-17-one 5␣-androstane-17␤-ol-3-one 5␤-pregnan-3␣-ol-20-one

Conc. (␮M)

rCBR3

rCBR3 W230P

hCBR3

hCBR3 P230W

CHCR3

CHCR3 W230P

200 25 25 25 25

100 17 n.d. 5.0 n.d.

100 12 n.d. 3.7 n.d.

100 17 n.d. 14 13

100 13 n.d. 9.0 8.5

100 2.3 n.d. 2.3 7.6

100 3.2 n.d. 2.2 8.7

Reductase activities toward steroids are shown as the ratio of reductase activity toward menadione. Each value represents the mean obtained from three experiments. S.D. of experiments was within 25%. n.d., no traceable activity was detected.

probably because the elongation of ␣G induced the additional hydrogen bond formed between the carbonyl group of Ser-240 and the amide group of Val-244 in the main chain following the substitution of Pro-244 (hCBR1) for Val-244 (hCBR3) [6,21]. In another hinge region of the substrate-binding loop, the PGW sequence at the carbonyl terminus of ␣F was highly conserved except in hCBR3 (PGP). Since Pro is a structurally rigid amino acid, the substitution appeared to affect the enzymatic characteristics of hCBR3. Therefore, we investigated the role of the residue by site-directed mutagenesis. The mutants exhibited properties similar to the parent enzymes with regard to steroid specificities and kinetics toward test substrates (Tables 2 and 3). Thus, the substitution alone had no apparent impact on the carbonyl reductase activities of CBR3. We revealed the importance of the substrate-binding loop region in CBR for the carbonyl reductase activities by the enzymatic characterization of chimera proteins between hCBR1 and hCBR3 (T. Miura, T. Nishinaka, T. Terada, unpublished results). Considering with the present results, the whole structure of the region is responsible for the carbonyl reductase activity, and the substitution of the amino acid residue at position 230 alone may be not sufficient to affect enzymatic properties. The crystal structure of hCBR3 is now available [21,22]. However, the substrate-binding loop structure in the crystal formed in a crystal contact [22], and therefore was unproductive. Additionally, it appears to be difficult to simulate the structure based on those of other SDR enzymes, since the loop is generally a flexible structure. Thus, docking studies between the enzyme and substrates could not be carried out. A productive form of the structure is needed for a further study of CBR3.

4. Conclusion We successfully cloned, expressed, and characterized rCBR3. We focused on and investigated the role of the amino acid residue at

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