Post-transcriptional analysis of rat mitochondrial D-3-hydroxybutyrate dehydrogenase control through development and physiological stages.

The nuclear encoded mitochondrial D-3-hydroxybutyrate dehydrogenase (BDH) is synthesized in the cytosal as a larger precursor. This membrane enzyme which requires lecithin for activity plays an essential role in energy metabolism as a ketone bodies-converting enzyme. A cDNA clone of the rat liver enzyme encompassing an antigenic determinant peptide has been isolated after immunoscreening of a lambda gt11 expression library. The nucleotide sequence of this 279-base cDNA insert contains a single open reading frame of 93 amino-acids, which represents about a third of the mature enzyme. Amino-acid sequence analysis predicts a hydrophobic stretch of 29 amino-acids long which probably functions as membrane anchor domain, or as an important region for the enzyme activation by phospholipid. By using this cDNA probe the BDH gene has been investigated at the mRNA level. There is only one mRNA (2-kb size) for BDH whatever the studied tissue. The rat gene is differently expressed since its mRNA is already present in the foetus liver while the BDH polypeptide amount is low and its enzymatic activity is not detectable even in the late stage of foetal development. The mRNA content is higher in the liver than in extrahepatic tissues. Adrenalectomy and ovariectomy increase liver mRNA content and polypeptide level, as well as activity of BDH. These effects are totally or partially abolished by corticosterone and estradiol treatments respectively. In addition, a 15-day hyperlipidic diet stimulates BDH gene expression. Present results show that the gene expression of this mitochondrial enzyme is modulated through development and hormonal and metabolic conditions mentioned above.     

Primary structure of rat liver D-beta-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase.

The amino acid sequence of D-beta-hydroxybutyrate dehydrogenase (BDH), a phosphatidyl-choline-dependent enzyme, has been determined for the enzyme from rat liver by a combination of nucleotide sequencing of cDNA clones and amino acid sequencing of the purified protein. This represents the first report of the primary structure of this enzyme. The largest clone contained 1435 base pairs and encoded the entire amino acid sequence of mature BDH and the leader peptide of precursor BDH. Hybridization of poly(A+) rat liver mRNA revealed two bands with estimated sizes of 3.2 and 1.7 kb. A computer-based comparison of the amino acid sequence of BDH with other reported sequences reveals a homology with the superfamily of short-chain alcohol dehydrogenases, which are distinct from the classical zinc-dependent alcohol dehydrogenases. This protein family, initially discerned from Drosophila alcohol dehydrogenase and bacterial ribitol dehydrogenase, is now known to include at least 20 enzymes catalyzing oxidations of distinct substrates.     

Cloning and expression of a functional rat liver D-beta-hydroxybutyrate dehydrogenase-beta-galactosidase fusion protein in Escherichia coli.

A rat liver bacteriophage lambda expression library was probed using polyclonal antibodies raised to purified rat liver D-beta-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.     

Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.     

Expression of R-3-hydroxybutyrate dehydrogenase, a ketone body converting enzyme in heart and liver mitochondria of ruminant and non-ruminant mammals.

1. The properties of rat liver and bovine heart R-3-hydroxybutyrate dehydrogenase (BDH) have been extensively studied in the past 20 years, but little is known concerning the biogenesis and the regulation of this dehydrogenase over different species. 2. In addition, controversial results were often reported concerning the activity, the level and the subcellular location of this enzyme in ruminants. 3. BDH activity found in liver and kidney mitochondria from ruminants (cow and sheep) is low, while it is much higher in rat. 4. However, the enzyme activity is detected in microsomes and in cytosol of liver and of kidney cells from ruminants. These activities are not correlated to ketonaemia level. 5. Although low BDH activity is detected in liver mitochondria from ruminants; the bovine liver BDH gene seems to be translated since BDH can be immunodetected by using an antiserum raised against bovine heart BDH. 6. Beside this, the good cross-reactivity between heart BDH and liver BDH suggests their high level of homology in ruminants.     

Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutylicum gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of cell extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.     

Characterization of a novel 3-hydroxybutyrate dehydrogenase from <Emphasis Type="Italic">Ralstonia pickettii</Emphasis> T1

We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.     

新标志物BDH1用于肝细胞癌术后生存的预测价值分析

目的:检测肝细胞癌组织中3-羟基丁酸脱氢酶1 (3-Hydroxybutyrate Dehydrogenase 1,BDH1)BDH1的表达情况,探讨其与肝细胞癌患者临床病理特征和术后生存的关系。方法:选取135例肝细胞癌根治性手术患者,整理临床病理及随访资料,调取相应的存档石蜡组织标本切片,采用免疫组化SP方法检测BDH1的表达,对染色结果评分,结合临床病理及随访数据进行统计分析。结果:肝癌细胞中BDH1的阳性表达主要定位于胞浆,阳性表达率为87.4%(118/135),其中低表达占48.1%(65/135),高表达占51.9%(70/135)。BDH1的表达与肝癌细胞癌组织分化级别、肿瘤直径、肿瘤数目、微血管侵犯及TNM分期均显著相关(P0.05)。多因素分析显示BDH1表达、TNM分期、组织分化级别是影响肝癌术后患者预后的独立危险因素(P0.01),BDH1高表达者术后总生存率较低表达者显著升高。结论:BDH1可能作为预测肝癌预后的重要参考指标。     

Role of phospholipids in activation of mitochondrial D(-)-beta-hydroxybutyrate dehydrogenase

Although phosphatidylcholine (PC) has been shown to be the type of phospholipid required for activation of mitochondrial beta-hydroxybutyrate dehydrogenase (BDH), mixtures of phospholipids containing PC are more effective activators. This study shows that apo-BDH, purified from bovine-heart mitochondria, and phospholipid-reconstituted BDH appear to be polydisperse. Upon cross-linking with dimethylpimelimidate and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the enzyme exhibited molecular weight forms from monomeric to heptameric BDH as well as higher molecular weight aggregates that did not much penetrate the gels. When different phospholipid mixtures containing PC were used to activate apo-BDH, and the reconstituted samples were subjected to cross-linking and SDS-gel electrophoresis, a direct relationship was found between the activating effect of the phospholipids used and BDH monomer concentration in the gels. The effectiveness order of phospholipids used was as follows: a mixture of PC, phosphatidylethanolamine and diphosphatidylglycerol in a molar ratio of 5:4:1 greater than bovine-heart mitochondrial phospholipids greater than Asolectin greater than PC. These results suggest the following. In addition to PC, which is required by BDH, other types of phospholipids play a role in activation of purified apo-BDH, possibly via enzyme disaggregation. The activity exhibited by purified, phospholipid-reconstituted BDH is associated mainly with the lower molecular aggregates of the enzyme, especially monomeric BDH.     

Prehibernation and hibernation effects on the D-3-hydroxybutyrate dehydrogenase of the heavy and light mitochondria from liver jerboa (Jaculus orientalis) and related metabolism

The D-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) from liver jerboa (Jaculus orientalis), a ketone body converting enzyme in mitochondria, in two populations of mitochondria (heavy and light) has been studied in different jerboa states (euthermic, prehibernating and hibernating). The results reveal: (1) important variations between states in terms of ketones bodies, glucose and lipid levels; (2) significant differences between the BDH of the two mitochondrial populations in term of protein expression and kinetic properties. These results suggest that BDH leads an important conformational change depending on the physiological state of jerboa. This BDH structural change could be the consequence of the lipid composition modifications in inner mitochondrial membrane leading to changes in BDH catalytic properties.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

Characterization of two D-beta-hydroxybutyrate dehydrogenase populations in heavy and light mitochondria from jerboa (Jaculus orientalis) liver

Mitochondrial membrane-bound and phospholipid-dependent D-beta-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a ketone body converting enzyme in mitochondria, has been studied in two populations of mitochondria (heavy and light) of jerboa (Jaculus orientalis) liver. The results reveal significant differences between the BDH of the two mitochondrial populations in terms of protein expression, kinetic parameters and physico-chemical properties. These results suggest that the beta-hydroxybutyrate dehydrogenases from heavy and light mitochondria are isoform variants. These differences in BDH distribution could be the consequence of cell changes in the lipid composition of the inner mitochondrial membrane of heavy and light mitochondria. These changes could modify both BDH insertion and BDH lipid-dependent catalytic properties.     

Human BDH2, an anti-apoptosis factor,is a novel poor prognostic factor for de novo cytogenetically normal acute myeloid leukemia

Background

The relevance of recurrent molecular abnormalities in cytogenetically normal (CN) acute myeloid leukemia (AML) was recently acknowledged by the inclusion of molecular markers such as NPM1, FLT3, and CEBPA as a complement to cytogenetic information within both the World Health Organization and the European Leukemia Net classifications. Mitochondrial metabolism is different in cancer and normal cells. A novel cytosolic type 2-hydroxybutyrate dehydrogenase, BDH2, originally named DHRS6, plays a physiological role in the cytosolic utilization of ketone bodies, which can subsequently enter mitochondria and the tricarboxylic acid cycle. Moreover, BDH2 catalyzes the production of 2, 3-DHBA during enterobactin biosynthesis and participates in 24p3 (LCN2)-mediated iron transport and apoptosis.

Results

We observed that BDH2 expression is an independent poor prognostic factor for CN-AML, with an anti-apoptotic role. Patients with high BDH2 expression have relatively shorter overall survival (P = 0.007) and a low complete response rate (P = 0.032). BDH2-knockdown (BDH2-KD) in THP1 and HL60 cells increased the apoptosis rate under reactive oxygen species stimulation. Decrease inducible survivin, a member of the inhibitors of apoptosis family, but not members of the Bcl-2 family, induced apoptosis via a caspase-3-independent pathway upon BDH2-KD.

Conclusions

BDH2 is a novel independent poor prognostic marker for CN-AML, with the role of anti-apoptosis, through surviving.     

Developmental regulation of D-beta-hydroxybutyrate dehydrogenase in rat liver and brain

The activities of ketone-metabolizing enzymes in rat brain increase 3- to 5-fold during the suckling period before decreasing to the adult level after weaning. We have observed that a similar developmental pattern also exists for D-beta-hydroxybutyrate dehydrogenase (BDH) in rat liver. Utilizing antibodies prepared against the purified protein we determined that the changes in BDH activities in both brain and liver are due to changes in the amount of BDH in the mitochondria. In vitro translations of isolated RNA followed by immunoprecipitation revealed that the increase in BDH activity and content was correlated with an increase in the level of functional BDH-mRNA in both liver and brain.     

Novel (2R,3R)-2,3-butanediol dehydrogenase from potential industrial strain Paenibacillus polymyxa ATCC 12321

A (2R,3R)-2,3-butanediol dehydrogenase (BDH99::67) from Paenibacillus polymyxa ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of meso- and (2S,3S)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which meso- and (2S,3S)-2,3-butanediol dehydrogenases belong.     

Two distinct alcohol dehydrogenases participate in butane metabolism by Pseudomonas butanovora

The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD(+)-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.