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Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis.

Mountassif D, Andreoletti P, El Kebbaj Z, Moutaouakkil A, Cherkaoui-Malki M, Latruffe N, El Kebbaj MS - BMC Biochem. (2008)

Bottom Line: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain.In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal.Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U866 (Institut National de la Santé et de la Recherche Médicale), Université de Bourgogne, LBMC (Biochimie Métabolique et Nutritionnelle), Faculté des Sciences, 6 Bd Gabriel, 21000 Dijon cedex, France. drissmountassif@yahoo.fr

ABSTRACT

Background: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions.

Results: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced.

Conclusion: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

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Alignment of BDH sequences. Alignment of BDH sequences from mammalian species (rat, mouse, human and jerboa) with Pseudomonas aeruginosa (P. a.) was realized using ClustalW (Thompson et al., 1994). Identical and similar residues were shown in black and yellow background respectively. The presumed amino acids sequences corresponding to oligonucleotides used for the PCR amplification of Jerboa BDH cDNA are underlined. According to the identity between Rat, Mouse and Human, they are considered as putative sequences in Jerboa. Amino acids of the catalytic tetrad Asn111, Ser139, Tyr152 and Lys156 (P. a. numbering) are marked by a star (*). These amino acids correspond to Asn114, Ser142, Tyr155 and Lys159 of the Pseudomonas fragi BDH (Ito et al., 2006). Amino acids participating to the NAD+ binding pocket Gly12, Leu61, Ala88, Ile90 and Ile108 (P. a. numbering) are marked by a hash sign (#). These amino acids correspond to Gly11, Leu64, Ala91, Ile93 and Leu113 of the Pseudomonas fragi BDH (Ito et al., 2006).
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Figure 2: Alignment of BDH sequences. Alignment of BDH sequences from mammalian species (rat, mouse, human and jerboa) with Pseudomonas aeruginosa (P. a.) was realized using ClustalW (Thompson et al., 1994). Identical and similar residues were shown in black and yellow background respectively. The presumed amino acids sequences corresponding to oligonucleotides used for the PCR amplification of Jerboa BDH cDNA are underlined. According to the identity between Rat, Mouse and Human, they are considered as putative sequences in Jerboa. Amino acids of the catalytic tetrad Asn111, Ser139, Tyr152 and Lys156 (P. a. numbering) are marked by a star (*). These amino acids correspond to Asn114, Ser142, Tyr155 and Lys159 of the Pseudomonas fragi BDH (Ito et al., 2006). Amino acids participating to the NAD+ binding pocket Gly12, Leu61, Ala88, Ile90 and Ile108 (P. a. numbering) are marked by a hash sign (#). These amino acids correspond to Gly11, Leu64, Ala91, Ile93 and Leu113 of the Pseudomonas fragi BDH (Ito et al., 2006).

Mentions: In order to clone the cDNA encoding BDH from jerboa liver, RT-PCR, primers were selected from two highly conserved BDH regions (LPGKALS and PMDYYWW) from mammalian species since the jerboa genome has not yet been sequenced. For the nucleotide sequence, see the section titled "Method" section. The amplification procedure revealed a single cDNA fragment with the expected size (936 pb) [Additional file 5]. The sequenced clone (GenBank accession # bankit 1072824 EU563473) was aligned and compared with other BDHs, from several species, including the mammalian vertebrate phyla and bacterial species, using the BioEdit program [36]. The highest identity was shown when the sequence was aligned with other mammalian BDH sequences (human, rat and mouse). Indeed, the analysis shown in Figure 2 reveals 79% identity with rat and mouse, 75% with human and only 19% with P. aeruginosa. Jerboa BDH sequence is 92% complete since amino acids from the C-terminal side are not yet available. The differences in sequences obtained between mammalian and bacterial BDHs can be related to the biochemical properties of both enzymes since mammalian BDH is membrane-bound and located in mitochondria and bacterial BDH is soluble and cytosolic. Moreover, the comparison between the two BDH types in terms of cDNA-deduced sequences reveals the major difference in the length of the polypeptide chain: 343 amino acids for the human BDH vs 256 for Pseudomonas. The longer sequence of the mammalian enzyme is related to the mitochondrial targeting presequence at the N-terminus and to the phospholipid-binding region at the C-terminus (Figure 2) [3,37]. The sequence alignment shows 48 identical amino acids and 42 similar amino acids between the mammalian and the bacterial enzymes.


Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis.

Mountassif D, Andreoletti P, El Kebbaj Z, Moutaouakkil A, Cherkaoui-Malki M, Latruffe N, El Kebbaj MS - BMC Biochem. (2008)

Alignment of BDH sequences. Alignment of BDH sequences from mammalian species (rat, mouse, human and jerboa) with Pseudomonas aeruginosa (P. a.) was realized using ClustalW (Thompson et al., 1994). Identical and similar residues were shown in black and yellow background respectively. The presumed amino acids sequences corresponding to oligonucleotides used for the PCR amplification of Jerboa BDH cDNA are underlined. According to the identity between Rat, Mouse and Human, they are considered as putative sequences in Jerboa. Amino acids of the catalytic tetrad Asn111, Ser139, Tyr152 and Lys156 (P. a. numbering) are marked by a star (*). These amino acids correspond to Asn114, Ser142, Tyr155 and Lys159 of the Pseudomonas fragi BDH (Ito et al., 2006). Amino acids participating to the NAD+ binding pocket Gly12, Leu61, Ala88, Ile90 and Ile108 (P. a. numbering) are marked by a hash sign (#). These amino acids correspond to Gly11, Leu64, Ala91, Ile93 and Leu113 of the Pseudomonas fragi BDH (Ito et al., 2006).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572057&req=5

Figure 2: Alignment of BDH sequences. Alignment of BDH sequences from mammalian species (rat, mouse, human and jerboa) with Pseudomonas aeruginosa (P. a.) was realized using ClustalW (Thompson et al., 1994). Identical and similar residues were shown in black and yellow background respectively. The presumed amino acids sequences corresponding to oligonucleotides used for the PCR amplification of Jerboa BDH cDNA are underlined. According to the identity between Rat, Mouse and Human, they are considered as putative sequences in Jerboa. Amino acids of the catalytic tetrad Asn111, Ser139, Tyr152 and Lys156 (P. a. numbering) are marked by a star (*). These amino acids correspond to Asn114, Ser142, Tyr155 and Lys159 of the Pseudomonas fragi BDH (Ito et al., 2006). Amino acids participating to the NAD+ binding pocket Gly12, Leu61, Ala88, Ile90 and Ile108 (P. a. numbering) are marked by a hash sign (#). These amino acids correspond to Gly11, Leu64, Ala91, Ile93 and Leu113 of the Pseudomonas fragi BDH (Ito et al., 2006).
Mentions: In order to clone the cDNA encoding BDH from jerboa liver, RT-PCR, primers were selected from two highly conserved BDH regions (LPGKALS and PMDYYWW) from mammalian species since the jerboa genome has not yet been sequenced. For the nucleotide sequence, see the section titled "Method" section. The amplification procedure revealed a single cDNA fragment with the expected size (936 pb) [Additional file 5]. The sequenced clone (GenBank accession # bankit 1072824 EU563473) was aligned and compared with other BDHs, from several species, including the mammalian vertebrate phyla and bacterial species, using the BioEdit program [36]. The highest identity was shown when the sequence was aligned with other mammalian BDH sequences (human, rat and mouse). Indeed, the analysis shown in Figure 2 reveals 79% identity with rat and mouse, 75% with human and only 19% with P. aeruginosa. Jerboa BDH sequence is 92% complete since amino acids from the C-terminal side are not yet available. The differences in sequences obtained between mammalian and bacterial BDHs can be related to the biochemical properties of both enzymes since mammalian BDH is membrane-bound and located in mitochondria and bacterial BDH is soluble and cytosolic. Moreover, the comparison between the two BDH types in terms of cDNA-deduced sequences reveals the major difference in the length of the polypeptide chain: 343 amino acids for the human BDH vs 256 for Pseudomonas. The longer sequence of the mammalian enzyme is related to the mitochondrial targeting presequence at the N-terminus and to the phospholipid-binding region at the C-terminus (Figure 2) [3,37]. The sequence alignment shows 48 identical amino acids and 42 similar amino acids between the mammalian and the bacterial enzymes.

Bottom Line: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain.In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal.Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U866 (Institut National de la Santé et de la Recherche Médicale), Université de Bourgogne, LBMC (Biochimie Métabolique et Nutritionnelle), Faculté des Sciences, 6 Bd Gabriel, 21000 Dijon cedex, France. drissmountassif@yahoo.fr

ABSTRACT

Background: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions.

Results: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced.

Conclusion: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

Show MeSH
Related in: MedlinePlus