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PubMed2005

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

Amino Acid Motifs↗
PubMed1981

Purification and properties of D-beta-hydroxybutyrate dehydrogenase from Zoogloea ramigera I-16-M.

D(-)-beta-Hydroxybutyrate dehydrogenase was purified from Zoogloea ramigera I-16-M to electrophoretic homogeneity. The molecular weight of the enzyme as determined by Sephadex G-200 gel filtration was 112,000, and the monomer molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 28,000, indicating that the native enzyme is a tetramer with four identical subunits. The enzyme showed a pH optimum at 8.0 in the oxidation reaction, and a broad pH optimum (5.5-7.5) in the reduction reaction. The Km values for D(-)-beta-hydroxybutyrate and NAD in the oxidation reaction were 3.2 X 10(-4) M and 5.7 X 10(-5) M, respectively. The Km value for acetoacetate in the reduction reaction was 1.5 X 10(-4) M and that for NADH was 1.5 X 10(-5) M. Acetyl CoA, D-lactate, and 2-hydroxybutyrate were effective inhibitors for the oxidation of D(-)-beta-hydroxybutyrate. The enzyme was sensitive to the inhibitory actions of sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid) and HgCl2.

Electrophoresis, Polyacrylamide Gel↗
PubMed1976

Acetyl-CoA production and utilization during growth of the facultative methylotroph Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate.

In Pseudomonas AM1, conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase, an acetoacetate: succinate coenzyme A transferase (specific for succinyl-CoA) and an inducible beta-ketothiolase. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase has been partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant (ICT54) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant (ICT51), lacking 'malate synthase' activity has been isolated and its characteristics indicate that this activity is normally essential for growth, of Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C1 compounds. The growth properties of a revertant (ICT51R) and of a mutant lacking malyl-CoA lyase (PCT57) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by way of acetyl-CoA.

Acetate-CoA Ligase↗
PubMed1987

Effect of anticonvulsant drugs on gamma-hydroxybutyrate release from hippocampal slices: inhibition by valproate and ethosuximide.

The effects of some anticonvulsant drugs have been investigated on gamma-hydroxybutyrate release from rat hippocampal and striatal slices. Sodium valproate and ethosuximide inhibited the depolarization-evoked release of gamma-hydroxybutyrate induced by 40 mM K+. The IC50 values for these two drugs are in the concentration range of valproate and ethosuximide that exists in rat brain after administration of anticonvulsant doses to the animals. Trimethadione and pentobarbital are without significant effects. It can be concluded that the inhibition of gamma-hydroxybutyrate release, particularly that observed for hippocampus, might explain the protective effect of valproate and ethosuximide on gamma-hydroxybutyrate-induced seizures and perhaps on other kinds of epileptoid phenomenon.

Animals↗
PubMed1972

Effect of -hydroxybutyrate on the release of monoamines from the rat striatum.

A simple in vitro system was developed to study the effect of gamma-hydroxybutyrate on nerve cell depolarization-induced release of labelled DA and 5-hydroxytryptamine. The release of (3)H-dopamine formed in rat striatal slices incubated with (3)3H-tyrosine was followed. A three minute exposure to K+ (53.0 mM) caused a thirty-fold increase in the release of newly synthesized (3)H-dopamine. This K + -induced release was antagonized when gamma-hydroxybutyrate (1 mM) was present in the medium. Potassium (53.0 mM) increased (eighteen-fold) the release of (3)H-dopamine from striatal slices initially loaded by preincubation with (3)H-dopamine. The K + -induced release of this pool of DA was, however, not antagonized by gamma-hydroxybutyrate.Potassium (53.0 mM) also increased the release from striatal slices of (3)H-5-hydroxytryptamine (5-HT) newly synthesized from (3)H-tryptophan. This K + -induced release of 5-HT was also not inhibited by gamma-hydroxybutyrate. The ability of gamma-hydroxybutyrate to antagonize only the K + -induced release of newly formed DA may explain why this agent causes a rapid and selective increase in brain dopamine.

Animals↗
PubMed1976

The effect of feeding on oxygen consumption, RQ and plasma levels of glucose, FFA, and D-beta-hydroxybutyrate in newborn infants of diabetic mothers and small for gestational age infants.

Eight infants of strictly controlled diabetic mothers (IDM), 8 infants of gestational diabetic mothers (IGDM) and 6 small for gestational age infants (SGA) were studied before the first feeding and during an early feeding regimen. In IDMs and IGDMs continuous monitoring from 2 hours up to 7 1/2 hours after birth before feeding revealed no consistent changes of VO2 and RQ. The groups of infants were studied on 4 different occasions: (I) 2 to 16 hours, (II) 1 to 2 days, (III) 3 to 4 days, and (IV) 7 to 11 days. Prefeeding VO2-values were not significantly different between each of the groups, but mean RQ was higher in IGDMs than in IDMs. Age of the infant and prefeeding RQ were inversely correlated (r = -0.537, p less than 0.02). With increasing age and milk intake VO2 increased significantly in all groups. RQ decreased during the first 24 to 48 hours in all groups and rose thereafter with highest values at 7 to 11 days. Plasma levels of glucose, FFA, and D-beta-hydroxybutyrate were not significantly different between each of the group. The highest values for D-beta-hydroxybutyrate were found at 1-2 days when the lowest RQ values were also recorded. D-beta-hydroxybutyrate concentrations and RQ values (r = 0.648, p less than 0.001) were inversely correlated suggesting increasing oxidation of fat. Feeding resulted in a marked rise in RQ to values around unity, which preceded a distinct increase in VO2 that reached a maximum at 1 to 1 1/2 hour after the feed, then slowly returned to pretest values. The rise in VO2 was accompanied by an increase in rectal temperature (0.4 to 1.5 degrees C). VO2, RQ, and plasma levels of glucose, FFA, and D-beta-hydroxybutyrate, were almost identical for each of the groups. We suggest: 1) That differences in feeding practice is the most likely explantation for the discrepancy between reported values for VO2, RQ, and circulating substrates in normal and low birth weight newborns. 2) That the rise in VO2 during the neonatal period, caused by feeding, reflects the cost of growth.

Adult↗
PubMed1983

D-(-)-poly-beta-hydroxybutyrate in membranes of genetically competent bacteria.

D-(-)-Poly-beta-hydroxybutyrate is a constituent of the membranes and the cytoplasms of genetically competent Azotobacter vinelandii, Bacillus subtilis, and Haemophilus influenzae cells. Within each species the concentration of D-(-)-poly-beta-hydroxybutyrate in the membranes and cytoplasm correlates with transformability. Fluorescence analysis of the thermotropic lipid phase transitions in A. vinelandii and B. subtilis cells indicates that D-(-)-poly-beta-hydroxybutyrate forms an organized gel structure in the membranes which is very labile. The concentration of organized D-(-)-poly-beta-hydroxybutyrate in the membranes, which can be estimated from the intensity of its phase transition, can be used to assess the competence of a culture.

Azotobacter↗
PubMed1986

Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli.

The effects of competence-inducing treatments on the composition and organization of membrane lipids in Escherichia coli K-12, DH1, DH5, HB101, and RR1 were investigated for two widely used protocols in which transformability is developed at low temperatures in Ca2+ buffers. At stages during each procedure, the lipid compositions of the cells were determined, and the thermotropic lipid phase transitions were observed in whole cell culture by fluorescence assay with the hydrophobic probe N-phenyl-1-naphthylamine. Competence was evaluated by determining transformation efficiencies with plasmid pBR322 DNA. The competence-inducing procedures effected only slight changes in phospholipid compositions which did not correlate with transformability. However, the induction of competence was coincident with de novo synthesis and incorporation of poly-beta-hydroxybutyrate into the cytoplasmic membranes and with the appearance of a sharp lipid phase transition above physiological temperatures. Transformation efficiencies correlated with poly-beta-hydroxybutyrate concentrations and with the intensity of the new phase transition. Transformability, poly-beta-hydroxybutyrate synthesis and the new phase transition were not significantly affected by inhibition of protein synthesis with chloramphenicol or inhibition of respiration or ATP synthesis with azide, cyanide, arsenate, or 2,4-dinitrophenol; however, when poly-beta-hydroxybutyrate synthesis was inhibited with acetaldehyde, the new phase transition was not observed, and competence failed to develop. These studies suggest that genetic transformability in E. coli may be physiologically regulated.

Cell Membrane↗
PubMed1997

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene.

An extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(-)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.

Amino Acid Sequence↗
PubMed2000

Requirement for the enzymes acetoacetyl coenzyme A synthetase and poly-3-hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti on PHB cycle intermediates.

We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.

Acyltransferases↗
PubMed1961

Assay of poly-beta-hydroxybutyric acid.

Law, John H. (Harvard University, Cambridge, Mass.) and Ralph A. Splepecky. Assay of poly-beta-hydroxybutyric acid. J. Bacteriol. 82:33-36. 1961-A convenient spectrophotometric assay of bacterial poly-beta-hydroxybutyric acid has been devised. Quantitative conversion of poly-beta-hydroxybutyric acid to crotonic acid by heating in concentrated sulfuric acid and determination of the ultraviolet absorption of the produce permits an accurate determination of this material in quantities down to 5 mug. This method has been used to follow the production of poly-beta-hydroxybutyric acid by Bacillus megaterium strain KM.

Bacillus↗
PubMed1965

Decomposition of poly-beta-hydroxybutyrate by pseudomonads.

Delafield, F. P. (University of California, Berkeley), M. Doudoroff, N. J. Palleroni, C. J. Lusty, and R. Contopoulos. Decomposition of poly-beta-hydroxybutyrate by pseudomonads. J. Bacteriol. 90:1455-1466. 1965.-A number of aerobic pseudomonads capable of using poly-beta-hydroxybutyrate as sole source of carbon have been partially characterized. One was selected for special study and described as a new species, Pseudomonas lemoignei Delafield. This bacterium constitutively produces extracellular digestive enzymes that hydrolyze the polymer to a mixture of d-beta-hydroxybutyrate and the dimeric ester of this acid, as well as an intracellular "dimer hydrolase" which hydrolyzes the dimeric ester. The exoenzymes are excreted during growth, but mainly at its cessation. Metabolism of beta-hydroxybutyrate largely suppresses enzyme excretion and causes a disappearance of excreted enzyme.

Hydroxybutyrates↗
PubMed1987

Cellular incorporation of poly-beta-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure.

Under growth-limiting conditions or conditions which mediate genetic transformation, Escherichia coli and Azotobacter vinelandii incorporate poly-beta-hydroxybutyrate into their plasma membranes. Genetic transformation competence of both bacteria increased in proportion to the concentration of membrane poly-beta-hydroxybutyrate. The effects of this lipid polymer on membrane structure were investigated by freeze-fracture electron microscopy. Before poly-beta-hydroxybutyrate incorporation, freeze-fracture revealed a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membrane. As the cells incorporated the lipid polymer into the membrane, transformability developed and small semiregular plaques which possessed shallow particles were seen. These plaques grew in size and frequency as the membrane poly-beta-hydroxybutyrate concentrations and transformability increased.

Azotobacter↗
PubMed2001

Isolation of poly(3-hydroxybutyrate) (PHB)-degrading microorganisms and characterization of PHB-depolymerase from Arthrobacter sp. strain W6.

Microbial degraders of poly(3-hydroxybutyrate) (PHB) were isolated from soil. Arthrobacter sp. strain W6 used not only PHB as a carbon source, but also PHAs such as poly(3-hydroxybutyrate-co-[5%]3-hydroxyvalerate), poly(3-hydroxybutyrate-co-[14%]3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-[22%]3-hydroxyvalerate). PHB-depolymerase was purified to homogeneity from the culture broth of Arthrobacter sp. strain W6 by a procedure involving DEAE- and butyl-Toyopearl column chromatographies. The Mr of the enzyme was estimated to be about 47,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 8.5 and 50 degrees C, and was inhibited by phenylmethylsulfonyl fluoride, Hg2+, Ag+, and Pb2+.

Arthrobacter↗
PubMed2000

Mixed ruminal microbes of cattle produce isopropanol in the presence of acetone but not 3-D-hydroxybutyrate.

The objective was to evaluate the ability of mixed rumen microbes to synthesize isopropanol from acetone or 3-D-hydroxybutyrate. Rumen fluid from seven mature, nonpregnant, dry Holstein cows was incubated with starch or cellulose and additions of acetone, 3-D-hydroxybutyrate, or saline. Rumen fluid was analyzed for isopropanol after 0, 3, 6, and 9 h. No isopropanol was present in any sample at 0 h, and none was present in incubations containing saline or 3-D-hydroxybutyrate at any subsequent time. Incubations that included acetone produced small amounts of isopropanol from 0 to 3 and 3 to 6 h and significantly larger amounts from 6 to 9 h. With starch as the energy substrate, production from 6 to 9 h was 3.8 micromol/min per liter of rumen fluid and 3.7 micromol/min per liter with cellulose as the energy substrate; however, these values did not differ significantly. Mixed rumen microbes could synthesize isopropanol from acetone but not from 3-D-hydroxybutyrate, and rumen microbial metabolism of acetone was the likely source of plasma isopropanol seen in ketotic ruminants.

2-Propanol↗
PubMed1975

[Antagonistic effect of sodium hydroxybutyrate on several effects of aminoxyacetic acid].

A well-known protective effect of aminooxyacetic acid against thiosemicarbazide convulsions was confirmed; it was shown that a similar, although somewhat weaker activity, was exerted by sodium hydroxybutyrate. Surprisingly, the effect of aminooxyacetic acid was diminished by sodium hydroxybutyrate. Sodium hydroxybutyrate in combination with aminooxyacetic acid decreased the protective activity of the latter against thiosemicarbazide convulsions and diminished the extent of GABA accumulation characteristic of aminooxyacetic acid action. This effect is attributed to the competition between the aminooxyacetic acid, sodium hydroxybutyrate and GABA for alpha-ketoglutarate-GABA-transaminase and possible for the GABA-ergic receptor.

Acetates↗
PubMed1976

Inhibition of adenosine 3':k'-monophosphate accumulation white fat acids, lactate, and beta-hydroxybutyrate.

The large increase in cyclic AMP accumulation by rat white fat cells seen in the presence of lipolytic agents plus methylxanthines and adenosine deaminase was markedly inhibited by lactate. However, lipolysis was unaffected by lactate. Octanoate, hexanoate, heptanoate, and beta-hydroxybutyrate inhibited both cyclic AMP accumulation and lipolysis by rat fat cells. The mechanism by which these acids inhibit lipolysis differs from that for long chain fatty acids such as oleate. Oleate directly inhibited triglyceride lipase activity of homogenized rat adipose tissue. In contrast, octanoate, beta-hydroxybutyrate, and lacatate had no effect on triglyceride lipase activity. Hormone-stimulated adenylate cyclase activity of rat fat cell ghosts was inhibited by oleate and 4mM octanoate but not by 1.6 mM octanoate, heptanoate, hexanoate, beta-hydroxybutyrate or lactate. None of the acids affected the soluble protein kinase activity of rat adipose tissue. There was no stimulation by lactate, butyrate, beta-hydroxybutyrate, or octanoate of the soluble or particulate cyclic AMP antilipolytic action of a short chain acid such as octanoate or hexanoate was not accompanied by any drop in total fat cell ATP. The mechanism by which lactate lowers cyclic AMP but not lipolysis remains to be established.

Adenylyl Cyclases↗
PubMed1982

[Calcium-dependent liberation of gamma-hydroxybutyrate after depolarization of rat brain slices].

The release of gamma-hydroxybutyrate from preloaded Rat brain striatal slices was investigated. Potassium induced depolarisation caused an efflux of gamma-hydroxybutyrate in the incubation medium. This release is significantly decreased by a calcium free medium, containing magnesium salt and EGTA. Taking into account our previous results concerning synthesis, uptake and binding of gamma-hydroxybutyrate, this calcium-dependent release strongly suggest a possible role of gamma-hydroxybutyrate in neurotransmission in the C.N.S.

Animals↗