Publications

• Reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (GHB).

  Bravo D.T., Harris D.O., Parsons S.M.

  Several assays for gamma-hydroxybutyrate (4-hydroxybutyrate, GHB) have been developed based on the enzyme gamma-hydroxybutyrate dehydrogenase (GHB-DH). Enzymatic oxidation of GHB by NAD+ is coupled to diaphorase-mediated reduction of pro-dye to yield colored product. GHB-DH from Ralstonia eutropha ... >> More

  Several assays for gamma-hydroxybutyrate (4-hydroxybutyrate, GHB) have been developed based on the enzyme gamma-hydroxybutyrate dehydrogenase (GHB-DH). Enzymatic oxidation of GHB by NAD+ is coupled to diaphorase-mediated reduction of pro-dye to yield colored product. GHB-DH from Ralstonia eutropha was cloned and expressed as a stable fusion protein easily purified by affinity chromatography. Quantitative initial velocity and endpoint versions of the assay in solution are described. Michaelis-Menten parameters for oxidation of GHB and ethanol were estimated. A semi-quantitative "dipstick" version of the assay on paper also is described. Both solution endpoint and "dipstick" assays are sensitive to about 0.05 mg GHB/mL using 10 microL of sample. Ethanol at concentrations possible in urine and agents used to stabilize physiological fluids for forensics analysis do not interfere significantly. The "dipstick" assay also allows detection of GHB in alcoholic beverages after evaporation of about one-fourth drop of beverage before testing. The enzymatic assay for GHB is reliable, sensitive, inexpensive and rapid. << Less

  J Forensic Sci 49:379-387(2004) [PubMed] [EuropePMC]

• Kinetics aspects of gamma-hydroxybutyrate dehydrogenase.

  Taxon E.S., Halbers L.P., Parsons S.M.

  Two groups of metabolically related enzymes, the Group III family of Fe²⁺-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state in ... >> More

  Two groups of metabolically related enzymes, the Group III family of Fe²⁺-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD⁺ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe²⁺ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed. << Less

  Biochim. Biophys. Acta 1868:140376-140376(2020) [PubMed] [EuropePMC]

• Enzymatic utilization of gamma-hydroxybutyric acid.

  NIRENBERG M.W., JAKOBY W.B.

  J Biol Chem 235:954-960(1960) [PubMed] [EuropePMC]

• Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli.

  Saito N., Robert M., Kochi H., Matsuo G., Kakazu Y., Soga T., Tomita M.

  The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia col ... >> More

  The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to gamma-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to gamma-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel gamma-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. << Less

  J. Biol. Chem. 284:16442-16451(2009) [PubMed] [EuropePMC]