Ronald B. Cain

University of Kent, Canterbury, ENG, United Kingdom

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Publications (16)50.05 Total impact

  • L.D.L. Jenkins · K A Cook · R B Cain
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    ABSTRACT: Three bacterial strains have been isolated that differ in their ability to degrade polyethylene glycols (PEGs). Strains R and O showed a marked preference for growth on the low and high molecular weight PEGs, respectively, while strain Z utilized mono-ethylene glycol only. The partial degradation of PEG 200 by strains R and O was studied in some detail and the results suggested that those components of the mixture that were not utilized were converted into acidic derivatives which accumulated in the medium.
    No preview · Article · Sep 1979 · The Journal of applied bacteriology
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    LESLEY D. L. JENKINS · K. A. COOK · R. B. CAIN
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    ABSTRACT: Three bacterial strains have been isolated that differ in their ability to degrade polyethylene glycols (PEGs). Strains R and O showed a marked preference for growth on the low and high molecular weight PEGs, respectively, while strain Z utilized mono-ethylene glycol only. The partial degradation of PEG 200 by strains R and O was studied in some detail and the results suggested that those components of the mixture that were not utilized were converted into acidic derivatives which accumulated in the medium.
    Preview · Article · Jul 1979 · Journal of Applied Microbiology
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    K. A. Cook · R. B. Cain
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    ABSTRACT: The immunoglobulin fraction of antiserum prepared against a homogeneous preparation of 3-carboxymuconate cyclase from Aspergillus niger ~6 was used to study the serological relatedness of the enzyme in crude extracts of other fungi. l'mmunodiffusion and immuno- electrophoresis experiments confirmed the homogeneity of the pure A. niger ~6 cyclase, its serological identity with the same enzyme in other strains of A. niger and the complete absence of any cross-reaction with the analogous 3-carboxymuconate cycloisomerase from Pseudomonas putida and Nocardia opaca. Extensive cross-reactions were observed with the cyclases from other species of Aspergillus and Penicillium. When such cross-reactions were examined quantitatively by the precipitin technique, several genera believed to be taxo- nomically related to Aspergillus and Penicillium on morphological grounds showed cross- reacting cyclases. Within the genera there was a broad correlation of serological relationships with morphological subdivisions. These correlations were considerably better than can be achieved by determinations of DNA base compositions because most of the genera examined have GC ratios of 50 to 55 mol %. Crude extracts of two taxonomically unrelated Basidio- mycetes showed no serological relatedness to the antisera.
    Preview · Article · Sep 1977 · Journal of general microbiology
  • David R. Thatcher · Ronald B. Cain
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    ABSTRACT: A preliminary investigation of the kinetic properties of 3-carboxy-cis,cis-muconate cyclase (EC 5.5.1.5) has been performed. The initial velocity of the reaction was shown to be proportional to the concentration of the enzyme in the assay system adopted and the apparent Km was found to be 57 muM at pH 6.0 and 30 degrees C but at concentrations exceeding 70 muM, substrate inhibition was apparent. At pH 6.0 the Ki for the substrate was 0.45 mM. Plots of V and Km against pH showed inflexions at pH 5.3 and pH 6.4. The enzyme was inhibited by a variety of inorganic anions and by a number of dicarboxylic and tricarboxylic acids. The degree of inhibition exerted by these acids was found to be proportional to the proximity of their carboxyl groups, the cis configuration being a more effective inhibitor than the trans configuration. As inhibition was competitive in each case, the presence of an anion-sensitive substrate-binding site has been postulated. The cis-cis, cis-trans and trans-trans isomers of muconate, 3-chloromuconate and 3-carboxy-cis-trans-muconate, close analogues of natural substrate but not attacked by the enzyme, were also found to be competitive inhibitors. The variation in pKi with pH was determined in the case of cis,cis-muconate and cis-aconitate, both of which gave curves suggesting the importance of a group with a pKa of approximately 6.4 responsible for increasing the inhibition of the enzyme. Modification by ethoxyformic anhydride and the kinetics of Rose-Bengal-sensitized photo-oxidation suggested the participation of a histidine residue in the catalytic reaction. These results are discussed in the light of recent work on enzymes catalysing analogous reactions; a likely reaction mechanism has been proposed.
    No preview · Article · Sep 1975 · European Journal of Biochemistry
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    ABSTRACT: Rhodotorula mucilaginosa was grown in p-hydroxybenzoate-limited chemostats over the dilution rate (D) range 0.01 to 0.17 h-1 and growth was adequately described by the Monod theory when maintenance energy requirements were considered. The p-hydroxy-benzoate affinity constant, K8, had the relatively high value of 270 mg/I. The yield from p-hydroxbenzoate varied with dilution rate but was constant above D equal 0.07 h-1 at 0.56 g yeast/g substrate utilised. The maintenance coefficeint for growth on the aromatic substrate was 20 mg/g yeast/hr. Culture viability decreased linearly as the dilution rate was reduced. 4-Hydroxybenzoate 3-mono-oxygenase, protocatechuate 3,4-dioxygenases, 3-carboxymuconate cyclase and 3-carboxymuconolactone hydrolase activities were dilution rate-dependent, results which accord with the substrate in inducibility of these enzymes. Under carbon-limited growth conditions the addition of glucose, a catabolite repressor of these enzymes, to the aromatic medium stimulated their synthesis. Data were also obtained which indicated that whereas the synthesis of the cyclase and the hydrolase was coordinately controlled, that of the first two enzymes of the 3-oxodipate pathway was under independent control.
    No preview · Article · Feb 1975 · Archives of Microbiology
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    G K Watson · R B Cain
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    ABSTRACT: 1. Two bacteria, a Bacillus sp. and a Nocardia sp. (strain Z1) were isolated from soil by enrichment with 0.1 percent (v/v) pyridine and grew rapidly on this compound as sole C, N and energy source. The monohydroxypyridines, tetrahydropyridine, piperidine and some other analogues were not utilized for growth or oxidized by washed suspensions of either bacterium. 2. Cell-free extracts were unable to metabolize pyridine even after supplementation with a variety of cofactors or protecting agents. Treatment of cells with toluene led to rapid loss of the ability to oxidize pyridine. 3. In the presence of 10mM-semicarbazide at pH 6.0, Nocardia Z1 accumulated a semialdehyde idenditied as its 2,4-dinitrophenylhydrazone by chromatography, mixed melting point, mass spectrometry and isotope trapping from [2,6(-14)C]pyridine as glutarate semialdehyde. 4. Extracts of this bacterium prepared from cells grown with pyridine or exposed to the gratuitous inducer 2-picoline, contained high activities of a specific glutarate semialdehyde dehydrogenase. 5. Cells grown with pyridine or glutarate also contained a glutaric dialdehyde dehydrogenase, an acyl-CoA synthetase and elevated amounts of isocitrate lyase but no glutaryl-CoA dehydrogenase. 6. Bacillus 4 accumulated in the presence of 10mM-semicarbazide several acidic carbonyl compounds from pyridine among which was succinate semialdehyde. Extracts of this bacillus after growth of the cells with pyridine contained an inducible succinate semialdehyde dehydrogenase in amounts at least 50-fold over those found in succinate-grown cells. 7. Two mutants of this bacillus, selected for their inability to grow on pyridine were deficient in succinate semialdehyde dehydrogenase. 8. In the presence of 0.2mM-KCN, washed suspensions of Bacillus 4 accumulated formate and possibly formamide from pyridine. The use of [14C]pyridine showed that formate was derived from C-2 of the pyridine ring. 9. The organism had a specific formamide amidohydrolase cleaving formamide quantitatively to formate and NH3. 10. Formate was further oxidized by the particle fraction. There was no soluble formate dehydrogenase in extracts.
    Preview · Article · Feb 1975 · Biochemical Journal
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    K A Cook · R B Cain

    Preview · Article · Dec 1974 · Journal of general microbiology
  • David R. Thatcher · Ronald B. Cain
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    ABSTRACT: 1The enzyme responsible for the conversion of 3-carboxy-cis-cis-muconate to 3-carboxymuconolactone in the fungus Aspergillus niger has been purified. This new purification method, utilizing ammonium sulphate fractionation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration produced a 530-fold purified preparation.2The purified enzyme appears to be homogeneous on dodecylsulphate-polyacrylamide disc gel electrophoresis and cellulose acetate electrophoresis.3A single major peak was observed in sedimentation velocity experiments and a s020,w of 8.75 S was calculated.4Calibrated gel filtration in a column of Sephadex G-200 suggested a molecular weight of approximately 200000 and a molecular radius of 4.63 nm. High-speed sedimentation equilibrium studies of this enzyme preparation indicated an apparent weight-average molecular weight of 191000. The frictional ratio of the enzyme was estimated as 1.22.
    No preview · Article · Nov 1974 · European Journal of Biochemistry
  • David R. Thatcher · Ronald B. Cain
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    ABSTRACT: 1The molecular weight of 3-carboxy-cis-cis-muconate cyclase obtained from Aspergillus niger has been determined under dissociating conditions. Equilibrium sedimentation studies in the presence of 5 M guanidine hydrochloride (0.1% v/v with respect to 2-mercaptoethanol) and dodecylsulphate-calibrated polyacrylamide gel electrophoresis suggested a minimum covalent molecular weight of approximately 24000 for the reduced enzyme.2Sedimentation equilibrium studies in the presence of a denaturant but in the absence of a reducing agent gave a value of 47000 for the molecular weight.3The amino acid and carbohydrate composition of the enzyme was determined and the presence of one thiol per subunit was shown to be available for titration with 5,5′-dithio-bis(2-nitrobenzoic acid).4The enzyme was digested with trypsin, and peptide-mapping experiments confirmed the hypothesis that 3-carboxy-cis-cis-muconate cyclase consists of eight subunits of approximately 24000 molecular weight and identical chemical composition.
    No preview · Article · Nov 1974 · European Journal of Biochemistry
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    G. Keith Watson · Charles Houghton · Ronald B. Cain
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    ABSTRACT: 1. Pyridine-3,4-diol (3,4-dihydroxypyridine, 3-hydroxypyrid-4-one), an intermediate in 4-hydroxypyridine metabolism by an Agrobacterium sp (N.C.I.B. 10413), was converted by extracts into 1mol of pyruvate, 2mol of formate and 1mol of NH(3) at pH7.0. 2. Formate, but not the alternative likely product formamide, was further oxidized fivefold faster by 4-hydroxypyridine-grown washed cells than by similar organisms grown on succinate. 3. The oxidation of pyridine-3,4-diol by crude extracts at pH8.5 required 1mol of O(2)/mol of substrate, produced 1mol of acid and led to the formation of formate and a new compound with an extinction maximum of 285nm (Compound I). This step was believed to be mediated by a new labile dioxygenase (t((1/2))=4h at pH7.0, 4 degrees C) cleaving the pyridine ring between C-2 and C-3. 4. Many of the properties of this pyridine-3,4-diol dioxygenase paralleled those of the extradiol (;meta') oxygenases of aromatic-ring cleavage. The extreme lability of the enzyme has so far precluded extensive purification. 5. Compound I showed changes in the u.v.-absorption spectrum with pH but after acidification it was converted into a new product, 3-formylpyruvate, with an extinction maximum now at 279nm. 6. Both Compound I and 3-formylpyruvate were metabolized by extracts but at very different rates. The slower rate of metabolism of Compound I was nevertheless consistent with that of pyridine-3,4-diol metabolism. 7. On acidification Compound I released about 0.65mol of NH(3) and has been identified as 3-formiminopyruvate. 8. 3-Formylpyruvate was hydrolysed to formate and pyruvate (K(m) 2mum) by an acylpyruvate hydrolase active against several other dioxo homologues. The activity of this enzyme was much lower in extracts of succinate-grown cells.
    Preview · Article · Jun 1974 · Biochemical Journal
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    J. Anthony Bird · Ronald B. Cain
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    ABSTRACT: 1. An organism isolated from sewage and identified as an Alcaligenes sp. utilized benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate as sole source of carbon and energy for growth. Higher alkylbenzenesulphonate homologues and the hydrocarbons, benzene, toluene, phenylethane and 1-phenyldodecane were not utilized. 2. 2-Phenylpropanesulphonate was metabolized to 4-isopropylcatechol. 3. 1-Phenylpropanesulphonate was metabolized to an ortho-diol, which was tentatively identified, in the absence of an authentic specimen, as 4-n-propylcatechol. 4. In the presence of 4-isopropylcatechol, which inhibited catechol 2,3-dioxygenase, 4-ethylcatechol accumulated in cultures growing on phenylethane-p-sulphonate. 5. Authentic samples of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 3-isopropylcatechol were oxidized by heat-treated extracts to the corresponding 2-hydroxyalkylmuconic semialdehydes. Ring cleavage occurred between C-2 and C-3. 6. The catechol derived from 1-phenylpropanesulphonate was oxygenated by catechol 2,3-dioxygenase to a compound with all the properties of a 2-hydroxyalkylmuconic semialdehyde, but it was not rigorously identified. 7. The catechol 2,3-dioxygenase induced by growth on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate showed a constant ratio of specific activities with catechol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol that was independent of the growth substrate. At 60 degrees C, activity towards these substrates declined at an identical first-order rate. 8. Enzymes of the ;ortho' pathway of catechol metabolism were present in small amounts in cells grown on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate. 9. The catechol 1,2-dioxygenase oxidized the alkylcatechols, but the rates and the total extents of oxidation were less than for catechol itself. The oxidation products of these alkylcatechols were not further metabolized.
    Preview · Article · Jun 1974 · Biochemical Journal
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    G. Keith Watson · Charles Houghton · Ronald B. Cain
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    ABSTRACT: 1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O(2) and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH(4))(2)SO(4) the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pK(m) versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.
    Preview · Article · Jun 1974 · Biochemical Journal
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    Ronald B. Cain · Charles Houghton · Keith A. Wright
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    ABSTRACT: 1. Washed suspensions of two Achromobacter species (G2 and 2L), capable of growth upon 2- and 3-hydroxypyridine respectively as sources of C and N, rapidly oxidized their growth substrate pyridine-2,5-diol (2,5-dihydroxypyridine) and the putative ring-cleavage product maleamate without a lag. Suspensions derived from fumarate plus (NH(4))(2)SO(4) cultures were unable to do so. 2. Extracts of both bacteria oxidized pyridine-2,5-diol with the stoicheiometry of an oxygenase forming 1mol of NH(3)/mol of substrate. 3. Heat-treated extracts, however, formed maleamate and formate with little free NH(3). 4. The conversion of maleamate into maleate plus NH(3) by extracts of strain 2L, fractionated with (NH(4))(2)SO(4), and the metabolism of maleamate and maleate to fumarate by extracts of both strains demonstrated the existence of the enzymes catalysing each reaction of the maleamate pathway in these bacteria. 5. The pyridine-2,5-diol dioxygenase (mol.wt. approx. 340000) in extracts of these Achromobacter species required Fe(2+) (1.7mum) to restore full activity after dialysis or treatment with chelating agents; the enzyme from strain 2L also had a specific requirement for l-cysteine (6.7mm), which could not be replaced by GSH or dithiothreitol. 6. The oxygenase was strongly inhibited in a competitive manner by the isomeric pyridine-2,3- and -3,4-diols.
    Preview · Article · Jun 1974 · Biochemical Journal
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    C Houghton · R B Cain
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    ABSTRACT: 1. Several species of micro-organisms that were capable of utilizing pyridine compounds as carbon and energy source were isolated from soil and sewage. Compounds degraded included pyridine and the three isomeric hydroxypyridines. 2. Suitable modifications of the cultural conditions led to the accumulation of pyridinediols (dihydroxypyridines), which were isolated and characterized. 3. Three species of Achromobacter produced pyridine-2,5-diol from 2- or 3-hydroxypyridine whereas an uncommon Agrobacterium sp. (N.C.I.B. 10413) produced pyridine-3,4-diol from 4-hydroxypyridine. 4. On the basis of chemical isolation, induction of the necessary enzymes in washed suspensions and the substrate specificity exhibited by the isolated bacteria, the initial transformations proposed are: 2-hydroxypyridine --> pyridine-2,5-diol; 3-hydroxypyridine --> pyridine-2,5-diol and 4-hydroxypyridine --> pyridine-3,4-diol. 5. A selected pyridine-utilizer, Nocardia Z1, did not produce any detectable hydroxy derivative from pyridine, but carried out a slow oxidation of 3-hydroxypyridine to pyridine-2,3-diol and pyridine-3,4-diol. These diols were not further metabolized. 6. Addition of the isomeric hydroxypyridines to a model hydroxylating system resulted in the formation of those diols predicted by theory.
    Preview · Article · Jan 1973 · Biochemical Journal
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    G K Watson · R B Cain

    Preview · Article · May 1972 · Biochemical Journal
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    C Houghton · G K Watson · R B Cain

    Preview · Article · Nov 1969 · Biochemical Journal

Publication Stats

256 Citations
50.05 Total Impact Points

Institutions

  • 1974-1979
    • University of Kent
      • Biological Laboratory
      Canterbury, ENG, United Kingdom
  • 1973-1974
    • Newcastle University
      Newcastle-on-Tyne, England, United Kingdom