3-Hydroxyisobutyrate dehydrogenase, an impurity in commercial 3-hydroxybutyrate dehydrogenase.
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ABSTRACT:
SUBMITTER: Worrall EB
PROVIDER: S-EPMC1147556 | biostudies-other | 1987 Jan
REPOSITORIES: biostudies-other
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Project description:D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.
| S-EPMC2664752 | biostudies-literature
Project description:We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a sigma54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.
| S-EPMC93451 | biostudies-literature
Project description:Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.
| S-EPMC3911200 | biostudies-literature
Project description:3-Hydroxybutyrate (3HB) is a product of interest as it is a precursor to the commercially produced bioplastic polyhydroxybutyrate. It can also serve as a platform for fine chemicals, medicines, and biofuels, making it a value-added product and feedstock. Acetogens non-photosynthetically fix CO2 into acetyl-CoA and have been previously engineered to convert acetyl-CoA into 3HB. However, as acetogen metabolism is poorly understood, those engineering efforts have had varying levels of success. 3HB, using acetyl-CoA as a precursor, can be synthesized by a variety of different pathways. Here we systematically compare various pathways to produce 3HB in acetogens and discover a native (S)-3-hydroxybutyryl-CoA dehydrogenase, hbd2, responsible for endogenous 3HB production. In conjunction with the heterologous thiolase atoB and CoA transferase ctfAB, hbd2 overexpression improves yields of 3HB on both sugar and syngas (CO/H2/CO2), outperforming the other tested pathways. These results uncovered a previously unknown 3HB production pathway, inform data from prior metabolic engineering efforts, and have implications for future physiological and biotechnological anaerobic research.
| S-EPMC9393629 | biostudies-literature
Project description:Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22) occupies a central position in central nervous system (CNS) neurotransmitter metabolism as one of two enzymes necessary for gamma-aminobutyric acid (GABA) recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1(-/-) knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2), represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH) or gamma-hydroxybutyrate (GHB; AKR7A2). A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE), an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE). Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue.
| S-EPMC2657722 | biostudies-literature
Project description:1,4-Butanediol (BDO)-used as solvent and abused for its euphoric effects-is converted to gamma-hydroxybutyrate (GHB) by the enzyme alcohol dehydrogenase. This double-blind, placebo-controlled crossover study with six healthy volunteers is the first to date investigating the role of the ADH inhibitor fomepizole (4-methylpyrazole (4MP)) in moderating this conversion in humans. Participants received on two different days either intravenous placebo or 15 mg/kg 4MP followed by oral administration of 25 mg/kg BDO. Pretreatment with 4MP resulted in significantly higher BDO maximal plasma concentration (P = 0.001) and area under the concentration-time curve (AUC; P = 0.028), confirming that ADH is the primary pathway for the conversion of BDO to GHB in humans. With 4MP, the mean arterial pressure was significantly lower at 105 minutes compared to baseline (P = 0.003), indicating that blood pressure lowering, observed not with a temporal relationship to 4MP administration but after the maximum BDO concentration was reached, may be an intrinsic effect of BDO.
| S-EPMC6465153 | biostudies-literature
Project description:D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energy-storage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD(+). An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C(3) atom of the substrate in the sp(3) configuration is positioned at a distance of 3.1 Å from the nicotinamide C(4) atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C(3). A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity.
| S-EPMC4933000 | biostudies-literature
Project description:Increased gamma-hydroxybutyric acid in urine and blood are metabolic hallmarks of succinic semialdehyde dehydrogenase deficiency, a defect of 4-aminobutyric acid metabolism. Here, we examined the hypothesis that succinic semialdehyde dehydrogenase deficiency could be identified via measurement of gamma-hydroxybutyric acid in newborn and post-newborn dried bloodspots. Quantitation of gamma-hydroxybutyric acid using liquid chromatography-tandem mass spectrometry in twelve archival newborn patient dried bloodspots was 360 ± 57 μM (mean, standard error; range 111-767), all values exceeding the previously established cutoff for newborn detection of 78 μΜ established from 2831 dried bloodspots derived from newborns, neonates and children. Gamma-hydroxybutyric acid in post-newborn dried bloodspots (n = 19; ages 0.8-38 years) was 191 ± 65 μM (mean, standard error; range 20-1218), exceeding the aforementioned GHB cutoff for patients approximately 10 years of age or younger. Further, gamma-hydroxybutyric acid in post-newborn dried bloodspots displayed a significant (p < .0001) inverse correlation with age. This preliminary study suggests that succinic semialdehyde dehydrogenase deficiency may be identified in newborn and post-newborn dried bloodspots via quantitation of gamma-hydroxybutyric acid, while forming the platform for more extensive studies in affected and unaffected dried bloodspots.
| S-EPMC6961708 | biostudies-literature
Project description:A recombinant form of D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) from Pseudomonas fragi has been crystallized by the hanging-drop method using PEG 3000 as a precipitating agent. The crystals belong to the orthorhombic group P2(1)2(1)2, with unit-cell parameters a = 64.3, b = 99.0, c = 110.2 A. The crystals are most likely to contain two tetrameric subunits in the asymmetric unit, with a VM value of 3.29 A3 Da(-1). Diffraction data were collected to a 2.0 A resolution using synchrotron radiation at the BL6A station of the Photon Factory.
| S-EPMC1952369 | biostudies-literature
Project description:Ketone bodies, including acetoacetate, 3-hydroxybutyrate, and acetone, are produced in the liver of animals during glucose starvation. Enzymes for the metabolism of (R)-3-hydroxybutyrate have been extensively studied, but little is known about the metabolism of its enantiomer (S)-3-hydroxybutyrate. Here, we report the characterization of a novel pathway for the degradation of (S)-3-hydroxybutyrate in anaerobic bacteria. We identify and characterize a stereospecific (S)-3-hydroxylbutyrate dehydrogenase (3SHBDH) from Desulfotomaculum ruminis, which catalyzes the reversible NAD(P)H-dependent reduction of acetoacetate to form (S)-3-hydroxybutyrate. 3SHBDH also catalyzes oxidation of d-threonine (2R, 3S) and l-allo-threonine (2S, 3S), consistent with its specificity for β-(3S)-hydroxy acids. Isothermal calorimetry experiments support a sequential mechanism involving binding of NADH prior to (S)-3-hydroxybutyrate. Homologs of 3SHBDH are present in anaerobic fermenting and sulfite-reducing bacteria, and experiments with Clostridium pasteurianum showed that 3SHBDH, acetate CoA-transferase (YdiF), and (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) are involved together in the degradation of (S)-3-hydroxybutyrate as a carbon and energy source for growth. (S)-3-hydroxybutyrate is a human metabolic marker and a chiral precursor for chemical synthesis, suggesting potential applications of 3SHBDH in diagnostics or the chemicals industry. IMPORTANCE (R)-3-hydroxybutyrate is well studied as a component of ketone bodies produced by the liver and of bacterial polyesters. However, the biochemistry of its enantiomer (S)-3-hydroxybutyrate is poorly understood. This study describes the identification and characterization of a stereospecific (S)-3-hydroxylbutyrate dehydrogenase and its function in a metabolic pathway for the degradation of (S)-3-hydroxybutyrate as a carbon and energy source in anaerobic bacteria. (S)-3-hydroxybutyrate is a mammalian metabolic marker and a precursor for chemical synthesis and bioplastics, suggesting potential applications of these enzymes in diagnostics and biotechnology.
| S-EPMC10305046 | biostudies-literature