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Biomedical subjects

N C Bruce

Publications and source records attributed to N C Bruce.

At least 37 records · Page 2Linked to original sources
PubMed1999

The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase.

The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.

Amino Acid Sequence↗
PubMed1998

Crystallization and preliminary diffraction studies of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

Pentaerythritol tetranitrate (PETN) reductase of Enterobacter cloacae PB2, a flavoprotein involved in the biodegradation of the explosive PETN, ethylene glycol dinitrate (EGDN) and glycerol trinitrate (GTN), was purified from an overexpressing strain of E. coli and crystallized at 293 K using the sitting-drop vapour-diffusion method. Diffraction data can be seen at 1.8 A. The primitive orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular-replacement calculations have been performed using a search model based on Old Yellow enzyme.

Bacterial Proteins↗
PubMed1998

Reductive and oxidative half-reactions of morphinone reductase from Pseudomonas putida M10: a kinetic and thermodynamic analysis.

The reaction of morphinone reductase (MR) with the physiological reductant NADH and the oxidizing substrate codeinone has been studied by multiple and single wavelength stopped-flow spectroscopy. Reduction of the enzyme with NADH proceeds in two kinetically resolvable steps. In the first step, the oxidized enzyme forms a charge-transfer intermediate with NADH. The charge-transfer complex is characterized by an increase in absorbance at long wavelength (540 to 650 nm), and its rate of formation is dependent on substrate concentration and is controlled by a second-order rate constant of 4. 8 x 10(5) M-1 s-1 at pH 7.0 and 5 degrees C. In the second step, the enzyme-bound flavin is reduced to the dihydroflavin form. The rate of flavin reduction (23.4 s-1 at pH 7.0 and 5 degrees C) is independent of substrate concentration and is observed as a monophasic decrease in absorbance at 462 nm. The oxidative half-reaction proceeds in three kinetically resolvable steps. The first is due to the formation of a reduced enzyme-codeinone charge-transfer complex and is observed at long wavelength (about 650 nm). The rate of charge-transfer complex formation is dependent on codeinone concentration and is controlled by a second-order rate constant of 11.5 x 10(3) M-1 s-1 at pH 7.0 and 5 degrees C. The second step represents flavin reoxidation and is observed at 462 (absorption increase) and 650 nm (absorption decrease) and progresses with a rate (about 45 s-1) which is independent of codeinone concentration. The third step is observed as a further small increase in absorbance at 462 nm and proceeds with a rate of about 2.5 s-1. This step most likely represents hydrocodone release from the oxidized enzyme. Analysis of the temperature dependence of the reductive half-reaction has enabled calculation of the entropic and enthalpic contributions for charge-transfer formation, charge-transfer decay (yielding free enzyme and substrate), and electron transfer to the enzyme-bound FMN, and the construction of a partial energy profile for the reaction catalyzed by MR. The reaction scheme and redox properties of MR are compared with those described previously for the closely related flavoprotein, old yellow enzyme. Although common features are identified, there are notable differences in the kinetic and redox properties of the two enzymes.

Anaerobiosis↗
PubMed1998

Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase.

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.

Aerobiosis↗
PubMed1998

The modified beta-ketoadipate pathway in Rhodococcus rhodochrous N75: enzymology of 3-methylmuconolactone metabolism.

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified beta-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

Adipates↗
PubMed1997

Crystallization and preliminary diffraction studies of morphinone reductase, a flavoprotein involved in the degradation of morphine alkaloids.

Morphinone reductase from Pseudomonas putida M10, a flavoprotein involved in the degradation of morphine alkaloids, was purified from an overexpressing strain of Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.5 A. The I-centred orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular replacement calculations have been performed using Old Yellow Enzyme as the search model.

Journal Article↗
PubMed1997

Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli.

The structural gene for heroin esterase was cloned from Rhodococcus sp. strain H1 and expressed in Escherichia coli BL21(DE3). The purified enzyme was found to be a tetramer with an M(r) of 137,000 and an apparent K(m) of 0.88 mM for 6-acetylmorphine. The G-x-S-x-G motif was observed in the deduced amino acid sequence, suggesting that the enzyme is a serin esterase.

Acetylesterase↗
PubMed1996

Bioluminescent assay for heroin and its metabolites.

Illicit heroin is trafficked as a solid particulate drug, while heroin abuse is monitored by testing urine samples for its principal metabolites, morphine and morphine-3-glucuronide. Two novel bacterial enzymes were used in the development of a linked-enzyme assay for heroin and its metabolites: heroin esterase, which converts heroin to morphine, and morphine dehydrogenase, which oxidizes morphine to morphinone with the concomitant reduction of NADP+. A bioluminescent assay involving heroin esterase, morphine dehydrogenase, and the bacterial luciferase from Vibrio harveyi was developed and shown to be sensitive to 89 ng/mL heroin and 2.0 ng/mL morphine. Excellent correlation with the results from 83 authentic samples submitted for urine drug screening at a hospital laboratory was obtained. The bioluminescent assay exhibited greater specificity and speed than current immunological screening methods. A novel format of the bioluminescent assay involving immobilized enzymes was sensitive to 101 ng (250 pmol) of heroin and responded well to particulate heroin. This form of the test was sensitive enough to respond to one or two typical particles of illicit heroin.

Acetylesterase↗
PubMed1996

Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF.

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.

Benzoates↗
PubMed1996

Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2.

A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.

Aerobiosis↗