1 National Institute of Agrobiological Sciences, Kannondai 212, Tsukuba, Ibaraki 305-8602, 3 Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011 and 4 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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Abstract |
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Keywords: diacetyl reductase/D-erythrulose reductase/gene expression/human P34H protein/organ distribution
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Introduction |
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The enzyme has been suggested to play a physiological role in detoxifying a wide variety of -dicarbonyl compounds contained in foods, because
-dicarbonyl compounds such as diacetyl are good substrates for D-erythrulose reductase from chicken liver (Maeda et al., 1998
). For the substrate specificity, D-erythrulose reductase was found to be similar to diacetyl reductases (EC 1.1.1.5) from pigeon (Diez et al., 1974
; Bernardo et al., 1984
) and bovine (Burgos et al., 1972; Provecho et al., 1984
) livers. This finding suggests that there is a structural relationship between the two different kinds of reductases from vertebrates. However, no amino acid sequences of these enzymes have been reported in the previous investigations.
This paper reports that the isolation and sequence analysis of a cDNA encoding the D-erythrulose reductase from chicken liver. Amino acid sequence analysis revealed that the D-erythrulose reductase is closely related to tetrameric carbonyl reductases in their structures. Although two enzymes with >60% similarity in the sequence are often annotated to the same proteins, we have found that this is not applicable for these two reductases. The results are discussed in terms of a comparison of the characteristics of D-erythrulose reductase with those of tetrameric carbonyl reductase.
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Materials and methods |
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Hybond-N+ (Amersham Pharmacia Biotech, Uppsala, Sweden) was used for screening and blotting. The ECL direct nucleic acid labeling and detection system was purchased from Amersham Pharmacia Biotech. Other chemicals were of the highest grade commercially available.
Purification of the enzyme
D-Erythrulose reductase was purified from chicken liver by the procedure of Maeda et al. (Maeda et al., 1998) with slight modifications. The enzyme was precipitated by using 5565% acetone fractionation, followed by centrifugation at 10 000 g for 30 min.
The enzyme activity was assayed by the measurement of the decrease in absorbance at 340 nm, due to consumption of NADPH, at 25°C in 0.1 M sodium phosphate buffer (pH 6.3). The test solution contained the enzyme preparation, 2.0 mM D-erythrulose and 50 nmol NADPH in a total volume of 1 ml. One unit of enzyme activity is defined as the reaction rate to oxidize 1 µmol of NADPH per minute.
Determination of partial sequences of the enzyme
Enzyme fragments digested by lysylendopeptidase were separated by reversed-phase HPLC (SMART System, Amersham Pharmacia Biotech). The digested solution was applied to a µRPC C2/C18 SC 2.1/10 column (Amersham Pharmacia Biotech) and eluted with a linear concentration gradient formed from 100 ml of 0.1% trifluoroacetate and an equal volume of 60% acetonitrile with 0.1% trifluoroacetate. The amino acid sequences of the purified fragments were determined by using an HP241 protein sequencer (Hewlett-Packard, Palo Alto, CA). The N-terminal sequence of the enzyme was also analyzed.
Preparation of DNA probe and cDNA library screening
A cDNA probe for D-erythrulose reductase was produced by reverse transcription polymerase chain reaction (RTPCR) from chicken liver mRNA (Clontech Laboratories, Palo Alto, CA). The RTPCR experiment was performed using a TaKaRa RNA PCR Kit Ver. 2.1 (TaKaRa Shuzo, Kyoto, Japan). Three degenerated primers were synthesized from peptide sequence information: primer 1, TCRTCNACYTCNGCRAAYTT; primer 2, GTNGTNATGACNGAYATGGG; and primer 3, GARGARYTNGTNMGNGARATG, corresponding to peptides 1, 2 and 3 (Figure 1). The characters R, Y, M and N signify A and T; C and T; A and C; and A, T, C and G, respectively. The PCR products were ligated into pCR2.1 vector by using original TA Cloning Kit (Invitrogen, Carlsbad, CA). Sequences of cloned DNA fragments were analyzed by using an automated DNA sequencer (ABI PRISM 310 Genetic Analyzer, PE Biosystems, Foster City, CA).
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Southern blot and northern blot analyses
Genomic DNA was extracted from chicken blood by using the TNEproteinase K method (Ivens et al., 1995). Following extraction with phenolchloroform, DNA was precipitated with 0.6 vol. of 2-propanol. Purified genomic DNA (5 µg) was digested with BglII, BamHI, EcoRI, EcoRV, HincII, HindIII, NdeI, SacI and SpeI. Digested DNA was separated by electrophoresis on 0.8% agarose gel and transferred to a nylon membrane by using 10xSSC (1.5 M NaCl and 0.15 M citrate). Hybridization was performed at 42°C in hybridization buffer [50 mM sodium phosphate buffer (pH 7.4) containing 0.7 M NaCl, 5 mM EDTA, 50% deionized formamide, 0.1% SDS, 0.1 mg/ml salmon sperm DNA and 5x Denhardts solution] supplemented with 32P-labeled NotINdeI fragment of cDNA clone of D-erythrulose reductase. cDNA probes were labeled using a random prime labeling kit (Wako Pure Chemical Industries, Osaka, Japan). The membranes were washed three times with 0.2x SSC and 0.1% NaCl at 65°C and exposed to an image analyzer (BAS-2000, Fuji Photo Film, Tokyo, Japan).
Total RNAs were extracted from the brain, heart, kidney, liver, lung, pancreas, spleen and testis of a 16-week-old male chicken using an RNeasy Midi Kit (QIAGEN, Hilden, Germany). Organs were frozen in liquid N2 and kept at 80°C until extraction. Frozen organs were homogenized using a micro homogenizer (Microtec, Funabashi, Japan), according to the manufacturers specifications. Extracted RNAs (10 µg) were denatured by the glyoxal method (McMaster and Carmichael, 1977), electrophoresed on 1.5% agarose gel in 10 mM sodium phosphate buffer (pH 7.0) and blotted on to a nylon membrane. Hybridizations were carried out in the same hybridization buffer as Southern blot analysis at 42°C overnight. Washing and image analysis were performed as described above.
Computational analyses
A BLAST (Altschul et al., 1990) search of the nr-aa (non-redundant amino acids) database was performed using the GenomeNet WWW server (http://www.genome.ad.jp/). The GAP program was used for pairwise alignment based on the method of Needleman and Wunsch (Needleman and Wunsch, 1970
) in the GCG program package (Genetic Computer Group/Accelrys, Madison, WI) version 10 in the MAFF DNA Bank (Maeda and Ugawa, 1998
). The 3D structure of carbonyl reductase (Tanaka et al., 1996
) (PDB entry = 1CYD) was visualized by Protein Adviser, ver. 3.5 (Fujitsu Kyushu System Engineering, Fukuoka, Japan). Comparative modeling was performed with MODELER (Molecular Simulations/Accelrys, San Diego, CA) based on the 3D structure of 1CYD. Calculation of the r.m.s.d. value was also performed with Protein Adviser, ver. 3.5.
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Results and discussion |
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Because D-erythrulose reductase was found to have a blocked N-terminal amino acid residue, we tried to remove the blocked N-terminal residues by assuming that it was formylamino acids, acetylserine/threonine and pyroglutamyl residues, but no signal was detected. We then digested the protein with lysylendopeptidase and separated peptide fragments by HPLC. The three following sequences of peptides 1, 2 and 3 were determined: AGARVTALSRTAADLESLVRECPGI, RVNTVNPTVVMTDMGRINW and FAEVDDVVNSILFLL.
The clone corresponding to a 1 kb fragment as the insert had a putative open reading frame encoding a protein of 246 amino acids (Figure 1). The amino acid sequence deduced from the DNA sequence contains the above three partial peptide sequences with a poly A sequence and an initiation methionine. The amino acid sequence contained the motif of short chain dehydrogenases/reductases family signature (PROSITE entry = PS00061), corresponding to Ser138Met165.
Expression pattern of the chicken D-erythrulose reductase
Expression of the D-erythrulose reductase in chicken organs was analyzed by northern blot analysis. Each amount of total mRNA was normalized by the amount of 18S rRNA (Figure 2B). In most organs, a signal corresponding to 1 kb was detected (Figure 2A
). The largest amount of mRNA was detected in the kidney and its distribution pattern was almost identical with that of enzyme activities in the other organs except for the relative amount in the testis (Figure 2A and C
).
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Sequence similarity search and evaluation of redundancy in homologous sequences
A BLAST search on the nr-aa (April 4, 2001) of GenomeNet showed that the chicken D-erythrulose reductase is similar to 10 sequences in the sequence databases, of which the e-values are less than 1e-50; ak007627_1, ak004023_1, af113123_1, 2517273a, 1920188a, cbr2_pig, cbr2_mouse, af090422_1, cbr2_caeel and ae003510_18. Figure 1 shows the multiple alignment of the chicken D-erythrulose reductase and the 10 similar sequences.
In the group of sequences, the pairs of ak004023_1 and ak007627_1 and af113123_1 and 2517273a have very high identity scores. Thus, redundancy of sequence should be discussed. Comparison of the amino acid sequences of ak007627_1 and ak004023_1 indicates that only one residue, the 224th, is different (see Figure 1). Comparison of two DNA sequences revealed that only one base (AGU of ak007627_1 to GGU of ak004023_1) is changed in the open reading frame and that the other residues are the same. Probably the difference is caused by single nucleotide polymorphisms (SNPs). Because the amino acid residue at position 224 is serine in all the sequences except ak007627_1 and ak004023_1, ak007627_1 was employed for calculation of the dendrogram. Regarding similarity, 98.9% of amino acid residues of af113123_1 and 2517273a are identical. The two differences are the 195th and 199th amino acid residues. The DNA sequence corresponding to 2517273a was not found in the GenBank/EMBL/DDBJ database (Okayama et al., 1998
; Benson et al., 2000
; Stoesser et al., 2001
). Comparison of the degenerated DNA sequence of 2517273a with the sequence of af113123 in GenBank indicates that two DNA residues were changed for each different amino acid residue (a total of four residues were different). Because it is difficult to determine that four substitutions in the sequence in humans are in the range of SNPs, these two sequences were handled independently in this analysis.
There are three sequences from mouse: ak007627_1, ak004023_1 and cbr2_mouse. As described above, ak007627_1 and ak004023_1 seem to be identical; however, cbr2_mouse is not (see the identity scores cbr2_mouse to ak007627_1 and ak004023_1 in Figure 4A). This score is comparable to that between the mouse carbonyl reductase (cbr2_mouse) and the human proteins (2517273a and af113123_1) (Figure 4a
). Therefore, it was concluded that cbr2_mouse and ak007627_1/sk004023_1 are independent proteins.
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Comparative modeling on the monomer form of the chicken D-erythrulose reductase based on the 3D structure of cbr2_mouse (PDB entry code = 1CYD) was performed. The r.m.s.d. value of corresponding C atoms between the modeled structure of D-erythrulose reductase and 1CYD was 0.21 Å. Next, atoms within 4 Å from heavy atoms of NADP+ and 2-propanol in 1CYD were determined (Figure 5
). In the multiple alignment of the D-erythrulose reductase (Figure 1
), the residues having any heavy atoms within a 4 Å distances from ligands are shown in blue, green and yellow columns (22 blue columns, around NADP+; two yellow columns, around 2-propanol; and three green columns, near both NADP+ and 2-propanol). When the sequence of the chicken D-erythrulose reductase was compared with that of the mouse tetrameric carbonyl reductase, most of the residues around the NADP+-binding site are identical except for Thr38 of the mouse carbonyl reductase. However, the two residues of the mouse carbonyl reductase surrounding 2-propanol, Val190 and Leu146, were replaced by Asn192 and His148 in the chicken D-erythrulose reductase, respectively (Figure 1
). These two residues are situated on the edge of the substrate pocket (Figure 5
).
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In the 3D structure of the mouse tetrameric carbonyl reductase (Tanaka et al., 1996), a total of 27 amino acids residues surround the two enzyme-bound ligands. NADP+ was adjacent to 25 residues (blue and green columns in Figure 1
) and 2-propanol was adjacent to five residues (yellow and green columns in Figure 1
). Three residues (green columns in Figure 1
) are neighbors to both ligands. Only one of the 25 amino acids around NADP+ in the carbonyl reductase was different from the D-erythrulose reductase (Thr38 of the carbonyl reductase corresponding to Ser40 of the D-erythrulose reductase). Because both threonine and serine have a hydroxyl group at the same distance from the
-carbon, the substitution may not alter the NADP+-binding pocket conformation.
Within the substrate binding pocket, two of five amino acids are different from the corresponding residues of the carbonyl reductase (Val190 to Asn192 and Leu146 to His148; the latter residues are contained in the sequence of the D-erythrulose reductase). The electrostatic fields in the pocket may change from hydrophobic to hydrophilic by the substitution. Because both asparagine and histidine have positive charges, polar compounds can make a more stable complex with D-erythrulose reductase than with carbonyl reductases. Although the mouse carbonyl reductase catalyzed the reduction of acetone as previously reported (Nakayama et al., 1988; Oritani et al., 1992
), D-erythrulose reductase did not reduce it. A change in electrostatic field in the active site would play an important role in such differences in their substrate selectivity.
Evolutionary consideration of the chicken D-erythrulose reductase and related proteins
As described above, eight independent proteins exhibit high similarity scores with respect to D-erythrulose reductase. The results of evolutionary analysis show that the proteins of vertebrates are separated into two groups (Figure 4B). One group (group I) comprises two tetrameric carbonyl reductases (cbr2_pig and cbr2_mouse) and hamster sperm P26h protein (af090422_1), and the other group (group II) includes chicken D-erythrulose reductase, human P34H protein (2517273a) and the other two proteins (af113123_1 and ak007627_1/ak004023_1). Because a divergence of vertebrates and others (fly and C. elegance) was detected in the dendrogram (Figure 4b
), it is presumed that the proteins of groups I and II were separated in the evolutionary process after this divergence. In addition, because two kinds of mouse proteins were reported and were distributed in groups I and II, these two groups of enzymes are assumed to be duplicated and differentiated before the beginning of the divergence of mammals.
Although two kinds of enzymes were known only in mouse, the existence in other animals is not clear. To determine the copy number and related sequences in chicken, genomic Southern analysis was performed. A major band was observed in the samples digested with EcoRI, HindIII, NdeI and SpeI. However, a minor band was also detected in the samples digested with HincII and SacI, suggesting the presence of possible related sequences (Figure 3). This result indicates that the gene encoding the D-erythrulose reductase is unique, but that there is a similar gene in chicken genome. The protein corresponding to tetrameric carbonyl reductase is unknown in chicken, but the second mRNA detected by the D-erythrulose reductase probe may code the chicken tetrameric carbonyl reductase.
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In the three groups of these proteins (group I, group II and non-vertebrate proteins), the functions of the non-vertebrate proteins (cbr2_caeel and ae003510_18) are not clear and the identities between the sequences of non-vertebrate proteins and those of the other proteins are <50% (Figure 4a). Especially, cbr2_caeel lacks a few residues (between Ala19 and Gly20) in the NADP+-binding site of cbr2_mouse (Figure 1
). Hence cbr2_caeel and ae003510_18 may be functionally different from vertebrate proteins (groups I and II) and also independent of each other.
On the other hand, group I and II proteins have conserved amino acid residues in their active sites, which suggests that they are NADP+-binding proteins. The residues around the substrate in the group I proteins and those predicted in the group II proteins are different (His148 to Leu and Asn192/Thr to Val; only the numbers of the D-erythrulose reductase are shown). This observation shows that the active sites around substrate in the group I proteins are relatively hydrophobic and those in the group II proteins are hydrophilic. Hence the group I proteins could prefer hydrophobic compounds and the group II proteins could select relatively hydrophilic compounds.
In the protein with unknown function, the residues in the active site of ak007627_1/ak004023_1 were almost identical with those of D-erythrulose reductase. The difference was Asp187 of the D-erythrulose reductase and Pro185 of ak007627_1/ak004023. This difference may cause the change in the affinity to NADP+ but not to the substrate.
The P26h (af0090422_1) protein and P34H (2517273a) are also unclear in function. Both of them are detected in the testis and their enzymatic functions have not been reported. Our analysis suggests that P26h protein is functionally close to carbonyl reductase (belonging to group I), whereas the P34H protein is close to D-erythrulose reductase (belonging to group II). Comparing the residues of their active site between P26h protein (af090422_1) and mouse tetrameric carbonyl reductase (cbr2_mouse), the differences are only from the 38th to the 40th residues. These residues may serve in the binding site of NADP+ and the substrate specificity of the P26h protein would remain as carbonyl reductase. Regarding the active site of the P34H protein (2517273a), there are two residues different from the D-erythrulose reductase: Ser185 from Asp187 and Thr190 from Asn192. Ser185 is located near NADP+ and Thr190 is near the substrate. Hence the P34H protein may exhibit a weak affinity for NADP+ and select relatively neutral substrates. However, Hosomi et al. detected the D-erythrulose reductase activity from human liver and the enzyme exists as a tetramer protein with a molecular mass similar to that of chicken (S.Hosomi, personal communication). If no other sequences are similar to D-erythrulose reductase in humans, the protein of 2517273a or af113123_1 annotated as human carbonyl reductase could be human D-erythrulose reductase.
Relationship among D-erythrulose reductases, dicarbonyl reductases and tetrameric carbonyl reductases
We previously reported the similarity of animal diacetyl reductases and D-erythrulose reductases (Maeda et al., 1998). Animal diacetyl reductases are also called dicarbonyl reductases because of the biochemical difference from bacterial diacetyl reductases. Although the sequences of dicarbonyl reductases are unknown, it is assumed that dicarbonyl reductases and D-erythrulose reductases are the same enzymes because their substrate specificities, inhibitor sensitivities and structural properties are very similar to each other. To distinguish animal diacetyl reductase from general diacetyl reductase and to show the similarity of animal diacetyl reductase and D-erythrulose reductase, this enzyme is called D-erythrulose/dicarbonyl reductase in the following paragraphs.
D-Erythrulose/dicarbonyl reductases and the tetrameric carbonyl reductases usually require NADPH as their coenzyme. These two kinds of reductases are structurally similar to each other: both of them are tetrameric enzymes and the similarity scores of amino acid sequences were >60% (Figure 4A). On the other hand, D-erythrulose/dicarbonyl reductases are enzymologically and physiologically distinguishable from tetrameric carbonyl reductases. Tetrameric carbonyl reductase is localized in mitochondria (Matsuura et al., 1994
) and has a free N-terminal methionine (Nakanishi et al., 1993
). The observations mean that in mitochondria a pre-protein has a signal peptide on the N-terminal site and the signal sequence is cleaved in the mature protein. On the other hand, D-erythrulose/dicarbonyl reductase is distributed in the cytosol of liver and kidney (as shown in Figure 2C
) and has a blocked N-terminal residue. This suggests that this enzyme has no transporting signal sequences at the N-terminus.
Previous reports showed that D-erythrulose/dicarbonyl reductase and tetrameric carbonyl reductase have had different substrate specificities (Nakayama et al., 1988; Maeda et al., 1998
; Oritani et al., 1992
). The activities for
-dicarbonyls are unclear about tetrameric carbonyl reductases. However, tetrameric carbonyl reductases catalyzed acetone and 4-nitrobenzaldehyde (Nakayama et al., 1988
; Oritani et al., 1992
), whereas D-erythrulose reductases did not (unpublished data). In the active site, two enzymes have 88% (23 of 26 residues) of identical residues within 4 Å from the ligands, but the substrate specificities of these enzymes are completely different with these few changes.
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Notes |
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Acknowledgments |
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References |
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Received May 21, 2001; revised March 25, 2002; accepted March 31, 2002.