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B K Barr

Publications and source records attributed to B K Barr.

7 recordsLinked to original sources
PubMed2000

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A.

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.

Actinomycetales↗
PubMed1998

Active-site binding of glycosides by Thermomonospora fusca endocellulase E2.

The determination of the high-resolution structure of the Thermomonospora fusca endocellulase E2 catalytic domain makes it ideal for exploring cellulase structure-function relationships. Here we present binding parameters (Kd, DeltaH degrees, and DeltaS degrees) describing the interaction of E2 with 4-methylumbelliferyl glycosides, determined by titrating the quenching of ligand fluorescence in equilibrium binding experiments. Quenched MU(Glc)2/E2 complexes were used as indicators in displacement titrations to measure the binding of natural glycosides and also of a nonhydrolyzable cellotetraose analogue. Binding of MU(Glc)2 and cellotriose were also determined by titration calorimetry. The results show that E2 binds glycosides exclusively in its active-site cleft, with high affinity and specificity. The observed patterns of ligand hydrolysis and the results with MU(Glc)2 as a substrate indicated that ligands bound to E2 with their nonreducing ends in position -2, consistent with the position of cellobiose in the E2cd structure. Polymerase chain reaction (PCR) mutagenesis of the conserved residue Tyr 73 (in E2 binding subsite -1) to Phe and Ser produced enzymes with lower activity but higher binding affinities, indicating that the volume of the subsite -1 binding pocket is crucial for enzyme function. Similarly, MUXylGlc (with its xylosyl unit located in position -1) bound with 100-fold higher affinity than MU(Glc)2. These results are similar to those for the related Trichoderma reesei exocellulase CBH II. The binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- and exocellulases.

Actinomycetales↗
PubMed1998

Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis.

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

Amino Acid Sequence↗
PubMed1996

Identification of two functionally different classes of exocellulases.

There are two classes of synergism in cellulase mixtures: synergism between endocellulases and exocellulases, and synergism between certain exocellulases. Exocellulases have been defined traditionally as releasing cellobiose from the nonreducing ends of cellulose, but this definition is inadequate to explain exo/exo synergism. Several recent reports indicate that some exocellulases are capable of hydrolyzing cellulose from the reducing end. The existence of two exocellulase classes with different specificities could provide an explanation for exo/exo synergism. In this paper, we report the substrate specificity of three Thermomonospora fusca (E3, E4, and E6) and two Trichoderma reesei (CBH I and CBH II) exocellulases on labeled cellooligosaccharides. We describe a new nonradioactive technique for determining substrate specificity, in which ion-spray mass spectrometry was used to analyze the products of enzymatic digests of cellopentaose labeled with 18O at the reducing end. Exocellulase reactivity was also investigated on cellopentaose labeled at the nonreducing end with 14C, and cellooligosaccharides reduced with NaBH4. The distribution of label in the reaction products supports the existence of two functional classes of exocellulases. One class (containing CBH I, E4, and E6) preferentially cleaves cellooligosaccharides from the reducing end, while the other (containing E3 and CBH II) preferentially cleaves from the nonreducing end. This classification of exocellulases is consistent with exo/exo synergism experiments, and with published cellulase crystallographic data.

Actinomycetales↗
PubMed1993

DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.

Actinomycetales↗