Biomedical subjects

R A Warren

Publications and source records attributed to R A Warren.

At least 19 recordsLinked to original sources

PubMed1995

Identification of the acid/base catalyst in Agrobacterium faecalis beta-glucosidase by kinetic analysis of mutants.

The catalytic mechanism of the retaining beta-glucosidase (Abg) from Agrobacterium faecalis involves a double-displacement process in which an alpha-glucosyl-enzyme intermediate is formed with general acid catalytic assistance and then hydrolyzed with general base assistance. Glu170 was identified as an important residue, possibly the acid/base catalyst, on the basis of sequence alignments. This glutamate is conserved in almost all enzymes in family 1 of glycoside hydrolases. Detailed pre-steady-state and steady-state kinetic analyses of the mutant E170G suggested very strongly that Glu170 is the acid/base catalyst. First, kcat values were invariant with pH over the range of 5.0-9.0. Secondly, rates of formation of the glycosyl-enzyme, calculated from kcat/Km and k2, were similar to those of wild-type enzyme for substrates not requiring protonic assistance but dramatically reduced for those needing acid catalysis. Thirdly, addition of azide as a competitive nucleophile increased kcat values 100-300-fold for substrates whose rate-limiting step is deglycosylation, yielding beta-glucosyl azide, but had no effect on the wild-type enzyme. Other anionic nucleophiles had similar, but less dramatic effects. Previous results [Gebler, J.C., et al. (1995) 34, 14547-14553] had indicated that Tyr298F is important for catalysis. The kinetic consequences of the mutations in the double mutant E170G-Y298F are additive, resulting in a 10(6)-fold reduction in kcat values and allowing the accumulation of a stable (t1/2 > 7 h) glucosyl-enzyme intermediate. Thus, Glu170 and Tyr298 function independently, and a possible role for Tyr298 in modulating the pKa of the catalytic nucleophile is proposed.

Amino Acid Sequence↗

PubMed1995

Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi.

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.

Actinomycetales↗

PubMed1995

The pTugA and pTugAS vectors for high-level expression of cloned genes in Escherichia coli.

Plasmids pTugA and pTugAS, designed for expression of cloned genes in Escherichia coli, possess the features of high-level inducible transcription, enhanced RNA translation, portability, high copy number, stability and versatility. In addition, pTugAS can be used to produce fusion proteins comprising a target protein and a cellulose-binding domain. Such fusion proteins can be purified in a single step by affinity chromatography on cellulose. Expression of two model gene fusions using the pTug plasmids resulted in yields of 500 mg of intracellular and 250 mg of extracellular recombinant protein per liter.

Alkaline Phosphatase↗

PubMed1995

Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop.

In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.

Amino Acid Sequence↗

PubMed1995

Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi.

The catalytic domains of beta-1,4-glucanases can be grouped into families of related amino acid sequences. The endoglucanase CenA from Cellulomonas fimi is a member of family B. All enzymes from this family are believed to hydrolyze beta-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond. Three-dimensional structures for two cellulases from family B have been determined by X-ray crystallographic analysis. These structures show that there are four Asp residues which are in a position to function as acid catalyst, base catalyst, and/or transition state stabilizers. These aspartates are conserved in all members of family B. The roles of Asp216, Asp252, Asp287, and Asp392, the corresponding amino acids in CenA, were determined. These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis, and the resulting effect on activity, substrate specificity, and overall structure has been determined. Changes in overall structure were monitored using circular dichroism spectroscopy, and no significant differences between the wild-type and mutant proteins were found. Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column. The Michaelis-Menten parameters of the enzyme were determined on 2,4-dinitrophenyl cellobioside as well as (carboxymethyl)-cellulose and phosphoric acid-swollen cellulose. Initial characterization of mutant proteins indicates that Asp252 and Asp392 are the acid and base catalysts, respectively, in CenA. Residue Asp287 appears to aid Asp252 in acid catalysis, and Asp216 is not absolutely required for catalysis.

Aspartic Acid↗

PubMed1995

Synaptic distribution of afferents from reticular nucleus in ventroposterior nucleus of cat thalamus.

This study was aimed at determining the synaptic circuitry that contributes to the alterations in thalamic function that accompany changes in behavioral states. The somatosensory sector of the thalamic reticular nucleus (RTN) was identified by microelectrode recording in cats and injected with Phaseolus vulgaris-leucoagglutinin (PHA-L). The axons of labeled RTN cells gave rise to collaterals within the RTN and continued into the dorsal thalamus where they terminated predominately in the ventral posterior lateral nucleus (VPL). After small injections in the upper limb representation of RTN, most labeled terminations in VPL were confined to its medial part, suggesting the presence of a topographic organization in the projection. Terminations were concentrated in localized, focal aggregations of boutons. Combined electron microscopic immunocytochemistry, using immunogold labeling for gamma-aminobutyric acid (GABA), showed that the PHA-L labeled boutons were GABA-positive terminals that ended in symmetrical synapses. Eighty-two percent of these synapses were on dendrites of relay neurons, 8.5% on dendrites of interneurons, and 9.3% on somata. The terminals of RTN axons form the majority of axon terminals ending in symmetrical synapses in VPL. Their concentration on relay neurons probably underlies the capacity of the RTN projection to reduce background activity of VPL relay neurons in the awake state and to maintain oscillatory behavior of these neurons in drowsiness and early phases of sleep.

Animals↗

PubMed1995

Purification of human interleukin-2 using the cellulose-binding domain of a prokaryotic cellulase.

Engineering gene fusions which introduce an affinity tag linked to the target polypeptide by a specific protease cleavage site is widely used to facilitate recombinant protein purification. A fusion protein CBDAPT-IL-2, comprised of the cellulose-binding domain (CBD) and Pro-Thr (PT) rich linker of the Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) and a factor Xa cleavage sequence (IleGluGlyArg) fused to the N terminus of human interleukin-2, was produced in Escherichia coli, Streptomyces lividans and mammalian COS cells. CBDAPT-IL-2, secreted from S. lividans or COS cells or recovered from the insoluble fraction of E. coli, could be purified by adsorption on cellulose. The intact fusion protein adsorbed to cellulose was hydrolyzed in situ with factor Xa to release active interleukin-2.

Actinomycetales↗

PubMed1994

Glutamate activation of cat thalamic reticular nucleus: effects on response properties of ventroposterior neurons.

The thalamic reticular nucleus (RTN) exerts an inhibitory influence upon the dorsal thalamus. During wakefulness and arousal, RTN neurons fire tonically, whereas during slow-wave sleep they fire rhythmic high frequency bursts. The effects produced by RTN inhibition upon the activity of dorsal thalamic neurons will therefore vary in relation to the firing mode of the RTN neurons. In the present study, we compared the effects of oscillating RTN neurons and of RTN neurons tonically activated with glutamate on the response profiles of single units reacting to controlled cutaneous stimulation in cat ventroposterior lateral thalamic nucleus (VPL). Experiments were performed under light barbiturate anesthesia and prior to the glutamate activation of the RTN, both RTN and VPL neurons showed spontaneous bursting patterns of activity consistent with the oscillatory mode. Typically, a cutaneous stimulus evoked a short latency excitatory response in VPL followed by a period of complete inhibition termed post-stimulus inhibition (PSI). In many neurons, the PSI was followed by a period of increased activity termed post-inhibitory excitation (PIE). Ejection of glutamate in the identified somatosensory division of the RTN shifted the oscillatory firing of its neurons to a high tonic mode and usually resulted in a decrease in VPL neuronal activity. Significant variations were observed in the occurrence and the magnitude of the effects among the different components of neuronal activity examined. Tonic activation of the RTN resulted in a significant reduction of ON- and OFF-PIEs in 81% of cases (30/37) and of spontaneous activity in 67% (22/33). In contrast, the response to a cutaneous stimulus was decreased in only 29% of cases (17/59) and was significantly increased in 24% (14/59). Tonic activation of the RTN by glutamate resulted in little change in the firing pattern of VPL neurons, and both short and long spike intervals were affected in a similar proportion. We conclude that the components of VPL neuronal activity most affected by switching RTN neurons from the oscillatory to the tonic mode are those normally dependent upon RTN neuronal oscillation. The present results also suggest that lowering background activity, such as occurs during the transition from sleep to wakefulness, is a factor leading to increase in the responsiveness of dorsal thalamic neurons.

Anesthesia↗

PubMed1994

A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose.

A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies. Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose. The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase. The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.

Biotin↗

PubMed1994

Oscillatory synaptic interactions between ventroposterior and reticular neurons in mouse thalamus in vitro.

1. The thalamic reticular nucleus (RTN) has reciprocal connections with relay neurons in the dorsal thalamus. We used whole cell recording in a mouse in vitro slice preparation maintained at room temperature to study the synaptic interactions between the RTN and the ventroposterior thalamic nucleus (VP) during evoked low-frequency oscillations. 2. After a single electrical stimulus of the internal capsule, postsynaptic potentials (PSPs) were recorded in all VP and RTN neurons. In 76% of slices, there was an initial response followed by recurrent PSPs lasting for up to 8 s and with a frequency of approximately 2 Hz in both the VP and RTN. 3. In RTN neurons the initial response consisted of a fast excitatory postsynaptic potential (EPSP) that generated a burst of action potentials. Recurrent PSPs consisted of barrages of EPSPs that often reached burst threshold. The structure of subthreshold EPSP barrages in RTN neurons suggested that they were generated by bursting VP neurons. 4. In VP neurons the stimulus usually evoked a small EPSP followed by a large inhibitory postsynaptic potential (IPSP) that was often followed by a rebound burst. This initial response was often followed by a series of recurrent IPSPs presumably generated by RTN bursts, because intrinsic inhibitory neurons are absent in rodent VP. 5. IPSPs in VP neurons and recurrent EPSPs in RTN neurons were completely abolished by application of a gamma-aminobutyric acid-A (GABAA) receptor antagonist. A GABAB receptor antagonist produced no or little change in either the initial or recurrent response. 6. Recurrent IPSPs in VP neurons were abolished by glutamate receptor antagonists before the initial IPSP, which always remained stimulus dependent. 7. The dependency of recurring IPSPs in VP and recurring EPSPs in RTN upon GABA-mediated inhibition and excitatory amino acid-mediated excitation, plus the character of recurring EPSPs in the RTN strongly suggest that the recurring events were generated through reverse-reciprocal synaptic interactions between VP and RTN neurons. These synaptic interactions most likely play an important role in thalamic oscillations in behavior.

Animals↗

PubMed1993

Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains.

The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed in Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface. Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays. In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added in the extracellular fluid. Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers. This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials. In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria.

Actinomycetales↗

PubMed1992

Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the bacterium Cellulomonas fimi.

Single crystals of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to better than 2.0 A resolution, belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have cell constants: a = b = 88.21 A, c = 81.10 A; alpha = beta = gamma = 90 degrees.

Actinomycetales↗

PubMed1992

Streptomyces lividans glycosylates an exoglucanase (Cex) from Cellulomonas fimi.

Exoglucanase Cex from Cellulomonas fimi is a glycoprotein [Langsford et al., J. Gen. Microbiol. 130 (1984) 1367-1376]. Cex produced by Streptomyces lividans from the cloned cex gene is also glycosylated. The extent and nature of glycosylation are similar for Cex from both organisms. The glycosylation affords protection against proteolysis for the enzymes from both organisms when they are bound to cellulose, but not in solution. The ability to glycosylate cloned gene products enhances the utility of Streptomyces as a host for the production of heterologous polypeptides.

Actinomycetales↗

PubMed1992

Mechanistic consequences of mutation of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase.

The replacement of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase by Asn and Gln by site-directed mutagenesis results in essentially complete inactivation of the enzyme, while replacement by Asp generates a mutant with a rate constant for the first step, formation of the glycosylenzyme, some 2500 times lower than that of the native enzyme. This low activity is shown to be a true property of the mutant and not due to contaminating wild-type enzyme by active site titration studies and also through studies of its thermal denaturation and of the pH dependence of the reaction catalyzed. Binding of ground-state inhibitors is affected relatively little by the mutation, while binding of transition-state analogues is greatly impaired, consistent with a principal role for Glu 358 being in transition-state stabilization, not substrate binding. Determination of kinetic parameters for a series of aryl glucosides revealed that the glycosylation step is rate determining for all these substrates in contrast to the native enzyme, where a switch from rate-limiting glycosylation to rate-limiting deglycosylation was observed as substrate reactivity was increased. These results coupled with secondary deuterium kinetic isotope effects of kH/kD = 1.17 and 1.12 measured for the 2,4-dinitrophenyl and p-nitrophenyl glucosides point to a principal role of the nucleophile in stabilizing the cationic transition states and in formation of the covalent intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparagine↗

PubMed1992

Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases.

Over 80 beta-1,4-glucanases and beta-1,4-xylanases can be classified into one of eight families on the basis of amino acid sequence similarities in their catalytic domains (Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991) Microbiol. Rev. 55, 303-315). As a test of this classification, the stereochemical course of hydrolysis of 10 enzymes representative of five families has been determined using proton NMR. These data, together with published data for six additional enzymes, show that representatives of a given enzyme family have the same stereoselectivity: four families catalyze hydrolysis with retention of anomeric configuration, two with inversion. The results support the hypothesis that family members share a common general fold, active site topology, and catalytic mechanism.

Endo-1,4-beta Xylanases↗

PubMed1992

Region-directed mutagenesis of residues surrounding the active site nucleophile in beta-glucosidase from Agrobacterium faecalis.

The active site nucleophile of the beta-glucosidase of Agrobacterium faecalis has recently been identified by the use of inhibitors. A combination of site-directed and in vitro enzymatic mutagenesis was carried out on the beta-glucosidase to probe the structure of the active site region. Forty-three point mutations were generated at 22 different residues in the region surrounding the active site nucleophile, Glu358. Only five positions were identified which affected enzyme activity indicating that only a few key residues are important to enzyme activity, thus the enzyme can tolerate a number of single residue changes and still function. The importance of Glu358 to enzymatic function has been confirmed and other residues important to enzyme structure or function have been identified.

Amino Acid Sequence↗

PubMed1992

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae.

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.

Amino Acid Sequence↗

PubMed1992

The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.

Adsorption↗