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R K Scopes

Publications and source records attributed to R K Scopes.

At least 19 recordsLinked to original sources
PubMed2000

Saccharococcus caldoxylosilyticus sp. nov., an obligately thermophilic, xylose-utilizing, endospore-forming bacterium.

Several closely related, xylanolytic, thermophilic bacilli were isolated from local soils on xylose-containing minimal medium. On the basis of morphology and biochemical characteristics, one of the isolates, designated strain S1812T (T = type strain), was studied further. Strain S1812T was a xylanolytic, sporulating, Gram-positive, rod-shaped bacterium. Its Gram-positive nature was confirmed by electron microscopic examination of thin sections of the cells. The isolate was a thermophilic (optimum temperature for growth, 65 degrees C), facultative anaerobe that grew on a wide range of carbon sources including glucose, lactose, starch and xylose. It expressed high levels of both xylose isomerase and xylulokinase on xylose and also on glucose. The DNA G + C content was 44 mol%. rRNA gene sequence analysis placed strain S1812T in Bacillus cluster 5; it was more closely related to Saccharococcus thermophilus than to thermophilic Bacillus species. DNA-DNA hybridization also indicated its close relationship to S. thermophilus. Based on the evidence presented, it is proposed that strain S1812T be designated Saccharococcus caldoxylosilyticus sp. nov. Strain S1812T is the type strain (= ATCC 700356T = DSM 97-987T).

Base Composition↗
PubMed1998

Sequence analysis and heterologous expression of the groE genes from Thermoanaerobacter sp. Rt8.G4.

The groE homologous genes of the anaerobic thermophile Thermoanaerobacter sp. Rt8.G4 (TRt) have been isolated, sequenced and analysed. The TRt groES and groEL encode subunits of chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) of 94 and 541 amino acids, respectively, and are arranged in that order forming the open reading frames (ORFs) of a bicistronic operon. A controlling inverted repeat of chaperone expression (CIRCE) element lies between the consensus promoter of the operon and TRt groES. At optimum growth temperature (65 degreesC) the chaperonins of TRt are expressed, but production of Cpn60 increases significantly following temperature increases of 3-10 degreesC. Functionally intact recombinant TRt chaperonins were produced in Escherichia coli. However, owing to codon incompatibility, replacement of consecutive AGA codons in the gene encoding TRt Cpn60 was necessary for optimum expression in this heterologous host.

Amino Acid Sequence↗
PubMed1998

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases.

The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degrees C), the mesophile Zymomonas mobilis (optimum growth temperature 32 degrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ.mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ.mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ.mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

Bacteria, Anaerobic↗
PubMed1998

Structure of the IgG-binding ligand of the T-gel.

We examine the ligand requirements for the divinylsulphone (DVS) based T-gel to bind immunoglobulins. The original gel consisted of 2-mercaptoethanol coupled to a DVS activated support, with both the thioether and sulphone sulphurs thought necessary for protein binding. No differences in the capacity for human IgG were observed for a highly activated gel coupled with mercaptoethanol, or when the same activated gel was incubated at high pH to hydrolyse the majority of its reactive groups before the remainder were coupled with the thiol, indicating that the thioether S may be replaced with a hydroxyl O. Increasing the time of the DVS-activation results in gels with higher concentrations of immobilised sulphone but lower concentrations of active groups. The IgG capacities of the mercaptoethanol coupled gels were found to increase with the time of the activation reaction, which may be exploited to produce high capacity gels while minimising the concentration of DVS. Reducing the vinyl of the DVS-activated gel with borohydride was found to decrease the amount of protein binding, with residual binding being attributed to the presence of hydrolysed or cross-linked sulphones in the gel. Reacting the activated gels with amines decreased the capacity for IgG still further, suggesting that not only are these ligands unable to bind IgG, they also prevent its interacting with neighbouring sulphones, perhaps due to the small amount of positive charge they carry.

Adsorption↗
PubMed1998

Thermostable variants of Zymomonas mobilis alcohol dehydrogenase obtained using PCR-mediated random mutagenesis.

Using a random mutagenesis technique, the ferrous-ion-activated alcohol dehydrogenase of Zymomonas mobilis has been altered to produce more thermally stable variants. After three rounds of mutation, a variant over 10 degrees C more stable at pH 8, with essentially unaltered kinetic characteristics, was produced. However, the pH profile of thermostability of this variant was much altered compared with the wild-type, with a relatively small increase (4 degrees C) at pH 6. Sequencing of the variants indicated five amino acids changes which contributed to thermostability: F9S, M13I, K31R, F90L, and G250D. Four of these were contained in the final stable variant, and the changes were partially additive, with individual mutations causing between 2 and 3.5 degrees C stability increases (at pH 7.5). It is estimated that the most stable variant would have a half-life under physiological conditions at 70 degrees C of 15 min.

Alcohol Dehydrogenase↗
PubMed1997

Ligand structure of the divinylsulfone-based T-gel.

The requirements for divinylsulfone (DVS)-based gels to act as thiophilic adsorbents, binding immunoglobulins in a salt-dependent manner have been examined. No differences in protein binding were observed for a DVS-activated gel reacted with mercaptoethanol (the T-gel), or for the same gel treated at high pH to hydrolyse the active groups and/or allow the formation of cross-links within the matrix, indicating that an O atom may be substituted for the thioether without affecting the thiophilic interactions. Extending the time of the activation reaction between DVS and the matrix results in increased amounts of sulfone attached to the gel, but decreased levels of active vinyl groups. When coupled to mercaptoethanol, these adsorbents bound more IgG than gels activated for shorter periods. This provides a convenient method to prepare thiophilic adsorbents of high capacity while minimising the amount of DVS used. The immobilised vinylsulfone must be linked to an electron donating atom for IgG to bind. When the vinyl was instead reduced with sodium borohydride, protein binding was decreased. No IgG bound to amine-coupled DVS-activated adsorbents, perhaps due to an overall positive charge on these gels at pH 7.4. The binding of human IgG to the adsorbents is dependent on ligand density, with little protein binding to gels having less than 16 mumol sulfone per ml. The binding increased with the ligand density above this level, with more than 25 mg IgG binding per ml to an adsorbent having 114 mumol sulfone per ml. The lack of binding at low ligand densities would be expected if the IgG must interact with two or more sulfone ligands to be retained on the adsorbent.

Borohydrides↗
PubMed1997

Allosteric control of Zymomonas mobilis glucose-6-phosphate dehydrogenase by phosphoenolpyruvate.

The second enzyme of the Entner-Doudoroff glycolytic pathway in Zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (PEP). In the presence of PEP levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a Hill coefficient up to 2. At low ionic strength in the absence of PEP, the response to glucose 6-phosphate concentration is Michaelis-Menten, but at physiological ionic strength and pH, a Hill coefficient of 1.3 to 1.4 was found even in the absence of PEP. Km values for NAD+ and NADP+ are also ionic-strength-dependent, increasing rapidly as salt concentration increases. Some sigmoidicity was also observed for NAD+ in the presence of PEP at low glucose 6-phosphate concentrations. The results can be interpreted in a Monod-Wyman-Changeux model, in which glucose 6-phosphate binds principally to the R-state, PEP to the T-state, and NAD+ to both states. These observations are clearly physiologically significant, and provide an explanation for the control of the balance between glycolytic throughput and ATP consumption in Z. mobilis.

Allosteric Regulation↗
PubMed1997

Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

Amino Acid Sequence↗
PubMed1997

Alteration of substrate specificity of Zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis.

Random mutagenesis of the gene encoding Zymomonas mobilis alcohol dehydrogenase-2 has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. After amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. Variants that are active on butanol have been investigated in detail. Changes at residue 161 and other changes at residues 155 and 165 cause enhanced activity with longer-chain alcohols. The 165 change also induces a marked alcohol-activation phenomenon that is observed not only with ethanol, but also with a nonsubstrate alcohol, 2-propanol, and with low concentrations of Triton X-100. These alterations to the alcohol binding pocket mainly introduce larger, more hydrophobic residues, suggesting that it is not the size but the hydrophobicity of the pocket that affects the substrate specificity. Variants active with NADP were isolated, and, as with similar variants of the yeast enzyme, they were found to have an Asp residue replaced by a neutral amino acid. However, unlike the yeast examples in which the affinity was substantially reduced, the affinity for NAD+ in these variants was little changed, and the affinity for NADP+ was higher than that for NAD+. As this enzyme is naturally ferrous ion-activated, and inactive with zinc, attempts were made to find variants that had activity with zinc. One was found, but the screening method also isolated other variants with altered metal ion preferences due to a mutation affecting amino acid 330.

1-Butanol↗
PubMed1996

The structure of glucose-fructose oxidoreductase from Zymomonas mobilis: an osmoprotective periplasmic enzyme containing non-dissociable NADP.

BACKGROUND: The organism Zymomonas mobilis occurs naturally in sugar-rich environments. To protect the bacterium against osmotic shock, the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) produces the compatible, solute sorbitol by reduction of fructose, coupled with the oxidation of glucose to gluconolactone. Hence, Z mobilis can tolerate high concentrations of sugars and this property may be useful in the development of an efficient microbial process for ethanol production. Each enzyme subunit contains tightly associated NADP which is not released during the catalytic cycle. RESULTS: The structure of GFOR was determined by X-ray crystallography at 2.7 A resolution. Each subunit of the tetrameric enzyme comprises two domains, a classical dinucleotide-binding domain, and a C-terminal domain based on a predominantly antiparallel nine-stranded beta sheet. In the tetramer, the subunits associate to form two extended 18-stranded beta sheets, which pack against each other in a face to face fashion, creating an extensive interface at the core of the tetramer. An N-terminal arm from each subunit wraps around the dinucleotide-binding domain of an adjacent subunit, covering the adenine ring of NADP. CONCLUSIONS: In GFOR, the NADP is found associated with a classical dinucleotide-binding domain in a conventional fashion. The NADP is effectively buried in the protein-subunit interior as a result of interactions with the N-terminal arm from an adjacent subunit in the tetramer, and with a short helix from the C-terminal domain of the protein. This accounts for NADP's inability to dissociate. The N-terminal arm may also contribute to stabilization of the tetramer. The enzyme has an unexpected structural similarity with the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD). We hypothesize that both enzymes have diverged from a common ancestor. The mechanism of catalysis is still unclear, but we have identified a conserved structural motif (Glu-Lys-Pro) in the active site of GFOR and G6PD that may be important for catalysis.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of a cobalt-substituted derivative of the iron-dependent alcohol dehydrogenase from Zymomonas mobilis.

The iron-dependent alcohol dehydrogenase from Zymomonas mobilis has been crystallized in a form suitable for X-ray diffraction studies. The crystals grew in hanging drops by vapor diffusion, equilibrating with a solution comprising 25-27% methoxypolyethylene glycol 5000 and 1 mM Co(2+) in a 0.2 M succinic acid/potassium hydroxide buffer at pH 5.5-5.7 at 281 K. Crystals are tetragonal, P4(1)22 (or P4(3)22), with unit-cell dimensions a = b = 125.7, c = 248.1 A. Four molecules comprise the asymmetric unit, and a self-rotation function indicates twofold local symmetry perpendicular to the unique axis and 15 degrees from a crystallographic twofold axis. Diffraction data to 3.0 A have been collected.

Journal Article↗
PubMed1996

Overexpression, purification, and generation of a thermostable variant of Zymomonas mobilis fructokinase.

The gene encoding fructokinase (EC 2.7.1.4) from Zymomonas mobilis has been expressed at high level in Escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4 degrees C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.

DNA Primers↗
PubMed1996

6-phosphogluconate dehydratase from Zymomonas mobilis: an iron-sulfur-manganese enzyme.

The enzyme 6-phosphogluconate dehydratase has been isolated in a stable form by a simple one-step procedure using dye ligand chromatography. The role of metal ions in the activity and stability of the enzyme was investigated. As with aconitase and several other dehydratase enzymes, the active site includes an Fe4S4 cluster. In addition, the purified enzyme has been shown to contain one manganese ion per subunit, which is also essential for activity. Rapid inactivation by superoxide radical was observed, which could only partly be protected by manganous ions The purified enzyme could be stabilised by alpha-glycerophosphate in place of manganese; glycerophosphate mimics the carbon atoms 4 to 6 of the natural substrate. This suggests that the manganous ion may involved in binding this part of the substrate.

Bacterial Proteins↗
PubMed1996

A simple and economical procedure for purification of muscle lactate dehydrogenase by batch dye-ligand adsorption.

A simple, economical and rapid purification procedure for muscle lactate dehydrogenase is described. It makes use of batch adsorption on Procion Red H-3B coupled to agarose through an amine linkage. The enzyme is eluted with NAD+ and sulphite, and for many purposes no more processing is necessary. Only 70 ml settled volume of adsorbent is required to process extract containing 1 g of lactate dehydrogenase, which is recovered with up to 90% yield, at over 98% purity. The method is readily scalable up or down, and makes an excellent class demonstration of affinity techniques.

Adsorption↗
PubMed1995

The single-ring Thermoanaerobacter brockii chaperonin 60 (Tbr-EL7) dimerizes to Tbr-EL14.Tbr-ES7 under protein folding conditions.

Chaperone proteins assist in the folding of some newly synthesized proteins and inhibit protein aggregation. The Thermoanaerobacter brockii chaperonin proteins (Tbr-EL and Tbr-ES) have recently been purified and characterized [Truscott, W.N., Høj, P. B., & Scopes, R. K. (1994) Eur. J. Biochem. 222, 277-284]; Tbr-EL was a single seven-membered toroid, unlike most GroELs which exist as double toroids. Using high-resolution gel filtration chromatography, we have resolved the purified Tbr-EL into single ringed (Tbr-EL7) and double ringed (Tbr-EL14) species. The latter contained tightly bound Tbr-ES co-chaperonin (Tbr-EL14.Tbr-ES7). In the presence of Mg.ATP and either Escherichia coli GroES (Eco-ES) or Tbr-ES (i.e., under protein folding conditions), the isolated Tbr-EL7 rapidly dimerized to the Tbr-EL14.Eco-ES7 or Tbr-EL14.Tbr-ES7 complexes. The doubly toroidal species thus formed contained > or = 6 molecules tightly bound ADP and one GroES7 and are similar to the asymmetric chaperonin complex isolated from Thermus thermophilus [Taguch, H., Konishi, J., Ishii, N., & Yoshida, M. (1991) J. Biol. Chem. 266, 22411-22418]. The isolated Tbr-EL7 and Tbr-EL14.Tbr-ES7 hydrolyzed ATP at approximate to 2 and 1 min-1, respectively. Addition of a molar excess of Eco-ES7 to the isolated Tbr-EL7 reduced the ATPase activity to 1 min-1, consistent with the formation of Tbr-EL14.Eco-ES7. Eco-ES7 failed to inhibit the Tbr-El14.Tbr-ES7 complex. The isolated Tbr-EL14.Tbr-ES7 complex did not support the folding of Rubisco under nonpermissive conditions. Only when the complex was supplemental with additional GroES was folding of Rubisco observed; i.e., one molar equivalent of GroES was not sufficient for folding. Both Tbr-EL7 and Tbr-EL14.Tbr-ES7 bound on unfolded [35S] Rhodospirillum rubrum Rubisco per mole particle. In contrast, Eco-EL14 bound 2 mol of protein per mole particle, consistent with each toroid having a peptide binding site. Eco-EL14.Eco-ES7 complex only bound one unfolded protein, thus GroES binding blocks one GroEL peptide binding site. Addition of Eco-ES7 to a Eco-EL14.Rubisco2 complex did not result in the displacement of one molecule of Rubisco but in the formation of a ternary Eco-EL14.Rubisco2.Eco-ES7 complex.

Adenosine Triphosphatases↗