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Biomedical subjects

H Dalton

Publications and source records attributed to H Dalton.

At least 55 records · Page 3Linked to original sources
PubMed1989

Cloning and expression in Escherichia coli of the toluene dioxygenase gene from Pseudomonas putida NCIB11767.

The genes encoding toluene dioxygenase, toluene cis-glycol dehydrogenase and catechol 2.3-oxygenase from Pseudomonas putida NCIB 11767 were cloned and expressed in Escherichia coli HB101 on a 20 kb fragment. The recombinant strain produced indigo and a variety of other coloured products. Although the enzymes were expressed in the absence of inducers, further induction was observed in the presence of toluene or benzene, implying the presence of regulatory elements on the 20 kb insert.

Cloning, Molecular↗
PubMed1988

The biosynthesis and assembly of protein A of soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Protein A of soluble methane monooxygenase (EC 1.14.13.25) of Methylococcus capsulatus (Bath) is the hydroxylase component of the enzyme complex, capable of inserting an atom of oxygen into methane. The protein possesses an unusual non-heme iron center consisting of two mu-hydroxo-bridged antiferromagnetically coupled iron atoms. It was possible to remove the iron center of protein A by subjecting the protein to freeze/thaw cycles or by dialysis against 8-hydroxyquinoline. Incubation of iron-depleted protein A with iron-EDTA and dithiothreitol resulted in the reassembly of the iron center of protein A as judged by restoration of enzyme activity (typically approximately 3 times the original activity) and by ESR spectroscopic methods. Reconstitution was inhibited by a number of metal ions, but none of the metals tested proved capable of replacing iron in the oxidation of methane. A number of iron-chelating agents exhibited weak inhibition of reconstitution. The KM(app) for iron in the reconstitution reaction was 133 microM. In vitro translation of total RNA isolated from M. capsulatus (Bath) produced protein A subunits equal to the native molecular weight, suggesting that there is no precursor form of this protein. Addition of the in vitro translated protein A to the reconstitution system resulted in low levels of hydroxylase activity suggesting that the subunits of protein A are self-assembling, a supposition supported by the presence of only one protein band on nondenaturing gels. Taken together, these data suggest that the subunits of protein A are synthesized in their native, mature form and self-assemble into an apoprotein A, into which the iron center is inserted.

Cations, Divalent↗
PubMed1987

Norfloxacin in the treatment of uncomplicated gonococcal infections.

Norfloxacin, an oral fluoroquinolone antibacterial, is active in vitro against a variety of gram-positive and gram-negative pathogens, including both penicillinase-producing and non-penicillinase-producing strains of Neisseria gonorrhoeae. An earlier study demonstrated that a two-dose regimen of norfloxacin was as effective as standard therapy with spectinomycin for treating gonococcal urethritis, including infections caused by penicillinase-producing organisms. In this randomized study of treatment for uncomplicated gonococcal infection in men and women, three oral treatment regimens were compared: patients received either two doses of norfloxacin (600 mg twice daily), a single dose of norfloxacin (800 mg), or a single-dose ampicillin (3.5 g)/probenecid (1.0 g) regimen (as recommended by the Centers for Disease Control). All three treatment regimens achieved similar cure rates. Although the number of patients treated was too small to yield statistically significant conclusions, it appears that norfloxacin may be slightly better treatment for rectal and pharyngeal gonococcal infections than ampicillin and probenecid. Additionally, norfloxacin was well tolerated in this study. Thus, based on a review of these data, norfloxacin appears to be an alternative, single-dose, oral treatment regimen for uncomplicated gonococcal infection.

Adult↗
PubMed1987

Purification and some properties of a novel heat-stable cis-toluene dihydrodiol dehydrogenase.

cis-Toluene dihydrodiol dehydrogenase was purified 200-fold from cells of a thermotolerant Bacillus species grown with toluene as the sole source of carbon and energy. The purified enzyme preparation was remarkably heat-stable and exhibited a half-life of 100 min at 80 degrees C, the temperature optimum. The activation energy of the reaction was 36 kJ.mol-1. Isoelectric focusing indicated that the pI of the native enzyme was 6.4 and that of the denatured enzyme 6.5. Although the pH optimum was 9.8, the enzyme was most stable at pH 8. The Mr of the enzyme was approx. 172,000 as determined by gel filtration and 166,000 by polyacrylamide-gel electrophoresis. The enzyme was composed of six apparently identical subunits with Mr values of 29,500. Kinetic analysis revealed that the Km for cis-toluene dihydrodiol was 92 microM and for NAD+ was 80 microM. The apparent Km values for cis-benzene dihydrodiol and cis-naphthalene dihydrodiol were 330 microM and 51 microM respectively. The enzyme was inhibited by mercurials but was unaffected by metal-ion chelators. Steady-state kinetics and product-inhibition patterns suggested that the enzyme mechanism was ordered Bi Bi.

Bacillus↗
PubMed1987

The effect of lipophilic weak acids on the segregational stability of TOL plasmids in Pseudomonas putida.

The effect of various lipophilic weak acids on the stability of certain TOL plasmids was investigated. Benzoate induced deletion of TOL plasmid DNA in Pseudomonas putida MT15, followed by loss of the plasmid; this effect was pH- and concentration-dependent, suggesting that undissociated benzoic acid was a more effective curing agent than the benzoate anion. Plasmid loss always approached a frequency of 100% after a lag and apparently depended on the prior occurrence of deletions, although deleted plasmid was stably maintained in the absence of the acid. m-Toluate, acetate and butyrate also induced deletions and plasmid loss at high frequencies, although these acids were less effective than benzoate. Benzoate inhibited the growth of plasmid-containing cells rather than permitting faster growth of cured cells on benzoate. Similar results were obtained with P. putida strains MT20 and MT84, which contain different TOL plasmids. We suggest that lipophilic weak acids induced deletions, possibly by excision of a transposon-like region, and disrupted the segregation of deleted plasmid.

Acetates↗
PubMed1986

Steady-state kinetic analysis of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath).

A steady-state kinetic analysis of purified soluble methane mono-oxygenase of Methylococcus capsulatus (Bath) was performed. The enzyme was found to follow a concerted-substitution mechanism. Methane binds to the enzyme followed by NADH, which reacts to yield reduced enzyme and NAD+. The reduced enzyme-methane complex binds O2 to give a second ternary complex, which breaks down to release water and methanol. In this way the enzyme can control the supply of electrons to the active site to coincide with the arrival of methane. Product-inhibition studies (with propylene as substrate) supported the reaction mechanism proposed. Ki values for NAD+ and propylene oxide are reported. The Km for NADH varied from 25 microM to 300 microM, depending on the nature of the hydrocarbon substrate, and thus supports the proposed reaction sequence. With methane as substrate the Km values for methane, NADH and O2 were shown to be 3 microM, 55.8 microM and 16.8 microM respectively. With propylene as substrate the Km values for propylene, NADH and O2 were 0.94 microM, 25.2 microM and 12.7-15.9 microM respectively. Methane mono-oxygenase was shown to be well adapted to the oxidation of methane compared with other straight-chain alkanes.

Alkanes↗
PubMed1986

The amino acid sequence of cytochrome c-555 from the methane-oxidizing bacterium Methylococcus capsulatus.

The amino acid sequence of the cytochrome c-555 from the obligate methanotroph Methylococcus capsulatus strain Bath (N.C.I.B. 11132) was determined. It is a single polypeptide chain of 96 residues, binding a haem group through the cysteine residues at positions 19 and 22, and the only methionine residue is a position 59. The sequence does not closely resemble that of any other cytochrome c that has yet been characterized. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50131 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment.

Amino Acid Sequence↗
PubMed1985

Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). A novel regulatory protein of enzyme activity.

An understanding of the mechanism of biological methane oxidation has been hampered by the lack of purified proteins. We describe here a purification protocol for the previously uncharacterized protein B of the soluble methane monooxygenase from the obligate methanotroph Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme consisting of a hydroxylase component, protein A, a reductase component, protein C, and protein B. All three proteins are required for monooxygenase activity. Protein B proves to be a low molecular weight (16,000) single subunit protein devoid of prosthetic groups. The protein is a powerful regulator of soluble methane monooxygenase activity, possessing the capacity to convert the enzyme from an oxidase to an oxygenase. Proteins A and C together catalyze the reduction of molecular oxygen to water, a reaction prevented by protein B. The uncoupling of soluble methane monooxygenase in this manner displays a number of novel features. First, the product of the uncoupled reaction is water, and second, the uncoupling is independent of substrate. Free hydrogen peroxide is not an intermediate in the reduction of oxygen by the incomplete methane monooxygenase enzyme complex. Finally, electron transfer can occur between protein C and protein A in the absence of protein B and protein B prevents the steady-state transfer of electrons in the absence of an oxidizable substrate, such as methane. It is demonstrated that oxygen reduction occurs at the active site of the hydroxylase component, protein A. A unifying mechanism, describing the interaction of the three proteins of soluble methane monooxygenase, is proposed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Copper(I), copper(II) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus capsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(II):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule. These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.

Binding Sites↗
PubMed1985

Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath).

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid-helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe-1 (oxidised), 1e-1 (semiquinone), 2e-1 (mostly semiquinone and reduced Fe2S2), and 3e-1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd-electron forms is consistent with its proposed role as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for passage to component A in one-electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.) The mid-point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD., Em = -150 mV; Fe2S2/Fe2.S2,Em = -220 mV; FAD./FAD..,Em = -260 mV. the presence of NADH did not alter these mid-point potentials. These mid-point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em = -320 mV) onto component A (Em = +150 mV and Em = -150 mV). The reducing power from NADH appears to be required by component A to activate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.

Binding Sites↗
PubMed1985

Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath).

Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.

Electrochemistry↗
PubMed1984

Purification and characterization of component A of the methane monooxygenase from Methylococcus capsulatus (Bath).

Methylococcus capsulatus (Bath) possesses a multi-component methane monooxygenase which catalyzes in vivo the conversion of methane to methanol. Component A of this enzyme system, believed to be the oxygenase component, has been purified to near homogeneity (95%). The native protein has a molecular weight of approximately 210,000 and is comprised of three subunits of Mr = 54,000, 42,000, and 17,000, which appear to be present in stoichiometric amounts suggesting an alpha 2, beta 2, gamma 2 arrangement in the native protein. Purified preparations of the protein are virtually colorless and examination of the uv/visible absorption spectrum revealed a peak around 280-290 nm and thereafter a steady decrease in absorbance to longer wavelengths. The ESR spectrum of the oxidized protein gave a signal at g = 4.3, presumably due to rhombic iron, and a radical signal at g = 2.01. Upon reduction with dithionite, a signal at g = 1.934 appeared. Chemical analyses of our purified preparations revealed the presence of iron (2.3 mol/mol) and zinc (0.2-0.5 mol/mol): molybdenum, copper, nickel, heme, and acid-labile sulfur were all virtually absent. On ultra thin layer isoelectric focusing, purified component A was judged to have a pI between 5.0 and 5.1. Extracts prepared from a variety of other methanotrophs failed to show any cross-reaction to antibody raised against M. capsulatus component A.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Purification of component A of the soluble methane monooxygenase of Methylococcus capsulatus (Bath) by high-pressure gel permeation chromatography.

A major improvement in the purification of the oxygenase protein (component A) of the methane monooxygenase has been effected. By employing high-pressure gel permeation chromatography several purification steps may be omitted from the previously published scheme. Furthermore the yield of the protein is enhanced and more importantly the recovered protein displays an increased specific activity, unlike that purified by other techniques.

Bacterial Proteins↗
PubMed1982

Co-metabolism.

There have been numerous instances reported when potentially recalcitrant compounds have been modified by microorganisms or completely mineralized by mixed communities or organisms; an example is pesticide biodegradation. Both situations rely upon the ability of microorganisms to transform compounds that they cannot utilize as sole sources of carbon and energy. This phenomenon of co-oxidation or co-metabolism has been fraught with confusion for many years as a result of the ambiguous use of terms and definitions. A redefinition of co-metabolism is proposed in an attempt to alleviate the problem: Co-metabolism--the transformation of a non-growth substrate in the obligate presence of a growth substrate or another transformable compound. The term 'non-growth substrate' describes compounds that are unable to support cell replication as opposed to an increase in biomass. This definition was devised primarily as a result of non-growth substrate metabolism studies with methane-utilizing bacteria. These studies are described in the text. The possible impact of endogenous polymer reserves on co-metabolic events is discussed. A number of examples where non-growth substrate metabolism is of environmental importance are presented, in particular the potential role of methane-oxidizing bacteria in the removal of CO from the environment. The evolutionary significance, if any, of fortuitous metabolism or co-metabolism is discussed, as are potential applications of these phenomena.

Bacteria↗
PubMed1979

Properties of the methane mono-oxygenase from extracts of Methylosinus trichosporium OB3b and evidence for its similarity to the enzyme from Methylococcus capsulatus (Bath).

1. The methane mono-oxygenase from Methylosinus trichosporium OB3b was soluble. The only suitable electron donor was NAD(P)H, neither sodium L-ascorbate nor electrons derived from the oxidation of methanol could substitute for NAD(P)H. Evidence is presented for the existence of an NAD+-linked formaldehyde dehydrogenase. 2. Mono-oxygenase activity was not inhibited by a range of potential inhibitors including potassium cyanide, amytal, carbon monoxide or various metal-chelating agents, although 8-hydroxyquinoline and ethyne were effective in this respect. 3. Although the enzyme preparations were unstable on storage, the crude extract could be resolved into two components by ion-exchange chromatography. Activity could be restored to one of the components on addition of purified components from Methylococcus capsulatus (Bath). 4. Cross-reactivity of mono-oxygenase components and other similarities between the enzymes from M. trichosporium and M. capsulatus are discussed. The properties of the M. trichosporium methane mono-oxygenase reported here are contrasted with the properties of the same enzyme reported by others.

Aldehyde Oxidoreductases↗