Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum

To elucidate the effects of C-terminal domains of LicMB (mature lichenase from Clostridium thermocellum) and terminal residues of LicMB-CD (catalytic domain of LicMB) on the properties of lichenase, a series of truncated genes were constructed and expressed in E. coli. The Thr-Pro box had a positive effect while the dockerin domain had a negative impact on the properties of LicMB. The N-terminal 10–25th and C-terminal 1–9th residues of LicMB-CD were necessary to retain high thermostability while the N-terminal 1–7th and C-terminal 1–3rd residues were not necessary to maintain enzymatic activity.     

Determination of immunological homology between cellulosome subunits and cloned endoglucanases and xylanase of Clostridium thermocellum

Immuno-cross reactivity between the subunits of Clostridium thermocellum cellulosome and cloned endogucanases and xylanase from the same bacterium was studied using the polyclonal antibodies raised against cloned enzymes. Dot blot analysis showed that the cellulosome, S8 and S11 subunits cross-reacted strongly with the antibodies of all cloned enzymes tested except that raised against CelC. Western blot analysis revealed that S8 and S11 subunits cross-reacted with the antibodies of CelA, CelB, CelD, CelG, CelH and XynZ, but the antibodies of CelB and CelG were highly specific for S8 and S11 subunits, respectively. Similar analysis using dissociated cellulosome showed that the antibodies of all cloned enzymes tested cross-reacted with more than one subunit of the cellulosome. Antibodies of CelC showed a very low cross-reactivity against all subunits of the cellulosome. The results indicate that immunological cross-reactivity studies could be useful, not only for demonstrating the similarities between native and cloned enzymes, but also for identifying native enzymes using antibodies of cloned enzymes.     

The sulfhydryls of firefly luciferase are not essential for activity

Firefly luciferase, containing an average of seven free sulfhydryls per two 50 000-dalton polypeptides, was modified by various sulfhydryl reagents. The differential reactivities of the sulfhydryls in luciferase protected by substrates allow one to define three categories of these groups: Class SH-III contains three sulfhydryls that are not involved in enzymatic activity. Class SH-II contains two sulfhydryls whose modification by different reagents causes varying effects on activity ranging from 0 to 60% inactivation. These sulfhydryls are not essential but may be important structurally or sterically. Class SH-I contains two sulfhydryls that are protected by substrates, either dehydroluciferyl adenylate or dehydroluciferin alone, and are located at or near the active site. The SH-I sulfhydryls are vicinal in the enzyme as demonstrated by their ability to form a disulfide bond. They have also been shown to exist on a single polypeptide chain. Modification of the SH-I groups by most reagents results in complete loss of enzymatic activity; reaction with methyl methanethiosulfonate produces an enzyme that emits only red light whereas native luciferase emits yellow-green light. Evidence is presented that the modified enzyme, while catalytically active, has a distorted active site. It is concluded that these two SH-I sulfhydryls are not essential for activity.     

Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Ebola virus VP40 late domains are not essential for viral replication in cell culture

Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.     

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.     

Two catalytic domains are required for protein deacetylation

Histone deacetylase (HDAC)-6 was recently identified as a dual substrate, possibly multisubstrate, deacetylase that can act both on acetylated histone tails and on alpha-tubulin acetylated on Lys40. HDAC-6 is unique among deacetylases in having two hdac domains, and we have used this enzyme as a useful model to dissect the structural requirements for the deacetylation reaction. In this report, we show that both hdac domains are required for the intact deacetylase activity of HDAC-6 in vitro and in vivo. The spatial arrangement of these two domains in HDAC-6 is essential and alteration of the linker region between the two domains severely affects the catalytic activity. Artificial chimeric HDACs, made by replacing the hdac domains in HDAC-6 with corresponding domains from other class II HDACs, show de novo deacetylase activity. Taken together, our results demonstrate for the first time that the spatial arrangement of hdac domains is critical for in vivo deacetylation reaction and may provide a useful model for the development of novel HDAC inhibitors.     

Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.     

Feruloyl esterase activity of the Clostridium thermocellum cellulosome can be attributed to previously unknown domains of XynY and XynZ

The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, beta-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungus Orpinomyces. Purified cellulosomes from C. thermocellum were found to hydrolyze FAXX (O-(5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D- xyl opyranosyl-(1-->4)-D-xylopyranose) and FAX(3) (5-O-[(E)-feruloyl]-[O-beta-D-xylopyranosyl-(1-->2)]-O-alpha-L- arabinofuranosyl-[1-->3])-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose) , yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia coli, and the expressed proteins hydrolyzed FAXX and FAX(3). The recombinant feruloyl esterase domain of XynZ alone (FAE(XynZ)) and with the adjacent cellulose binding domain (FAE-CBD(XynZ)) were characterized. FAE-CBD(XynZ) had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. K(m) and V(max) values for FAX(3) were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60 degrees C. PAX(3), a substrate similar to FAX(3) but with a p-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70 degrees C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount of p-coumaric acid. FAE(XynZ) had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAE(XynZ) resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the C. thermocellum cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.     

Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation

A full length cDNA for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, the membrane-bound glycoprotein that regulates cholesterol synthesis, was isolated from a human fetal adrenal cDNA library. The nucleotide sequence of this cDNA shows that the human reductase is 888 amino acids long and shares a high degree of homology with the hamster enzyme. The amino-terminal membrane-bound domain is the most conserved region between the two species (7 substitutions out of 339 amino acids). This region, which is predicted to span the endoplasmic reticulum membrane seven times, mediates accelerated degradation of reductase in the presence of sterols. The carboxyl-terminal catalytic domain is also highly conserved (22 substitutions out of 439 amino acids). However, the linker region between these two domains has diverged (32 substitutions out of 110 amino acids). Conservation of the structure of the membrane-bound domain in HMG-CoA reductase supports the hypothesis that sterol-regulated degradation is an important mechanism for suppression of reductase activity and for regulation of cholesterol metabolism in humans as well as in hamsters.     

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum.

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Cysteine residues are not essential for the catalytic activity of human class Mu glutathione transferase M1a-1a

To investigate the possible involvement of a Cys thiol in the catalysis of the human glutathione transferase M1a-1a, we constructed mutants of this enzyme wherein the four Cys residues present in the native enzyme were replaced by Ala residues. Three mutants, one where all four Cys residues had been replaced and two mutants where three out of four Cys residues were changed into Ala, were characterized regarding their catalytic activities with three different substrates as well as by their binding of three different inhibitors. All three Cys-deficient mutant forms of glutathione transferase M1a-1a were catalytically active with the tested substrates and their binding of inhibitors, measured by I50, were not significantly different from the values previously obtained for the wild-type enzyme. We therefore conclude that none of the Cys residues in this class Mu glutathione transferase are directly involved in the catalysis performed by this enzyme.     

Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum

Numerous bacterial and fungal organisms have evolved elaborate sets of modular glycoside hydrolases and similar enzymes aimed at the degradation of polymeric carbohydrates. Presently, on the basis of sequence similarity catalytic modules of these enzymes have been classified into 90 families. Representatives of a particular family display similar fold and catalytic mechanisms. However, within families distinctions occur with regard to enzymatic properties and type of activity against carbohydrate chains. Cellobiohydrolase CbhA from Clostridium thermocellum is a large seven-modular enzyme with a catalytic module belonging to family 9. In contrast to other representatives of that family possessing only endo- and, in few cases, endo/exo-cellulase activities, CbhA is exclusively an exocellulase. The crystal structures of the combination of the immunoglobulin-like module and the catalytic module of CbhA (Ig-GH9_CbhA) and that of an inactive mutant Ig-GH9_CbhA(E795Q) in complex with cellotetraose (CTT) are reported here. The detailed analysis of these structures reveals that, while key catalytic residues and overall fold are conserved in this enzyme and those of other family 9 glycoside hydrolases, the active site of GH9_CbhA is blocked off after the -2 subsite. This feature which is created by an extension and altered conformation of a single loop region explains the inability of the active site of CbhA to accommodate a long cellulose chain and to cut it internally. This altered loop region is responsible for the exocellulolytic activity of the enzyme.     

Conserved amino acid residues within the amino-terminal domain of ClpB are essential for the chaperone activity

ClpB from Escherichia coli is a member of a protein-disaggregating multi-chaperone system that also includes DnaK, DnaJ, and GrpE. The sequence of ClpB contains two ATP-binding domains that are enclosed between the amino-terminal and carboxyl-terminal regions. The N-terminal sequence region does not contain known functional sequence motifs. Here, we performed site-directed mutagenesis of four polar residues within the N-terminal domain of ClpB (Thr7, Ser84, Asp103 and Glu109). These residues are conserved in several ClpB homologs. We found that the mutations, T7A, S84A, D103A, and E109A did not significantly affect the secondary structure and thermal stability of ClpB, nor did they inhibit the self-association of ClpB, its basal ATPase activity, or the enhanced rate of the ATP hydrolysis by ClpB in the presence of poly-L-lysine. We observed, however, that three mutations, T7A, D103A, and E109A, reduced the casein-induced activation of the ClpB ATPase. The same three mutant ClpB variants also showed low chaperone activity in the luciferase reactivation assay. We found, however, that the four ClpB mutants, as well as the wild-type, bound similar amounts of inactivated luciferase. In summary, we have identified three essential amino acid residues within the N-terminal region of ClpB that participate in the coupling between a protein-binding signal and the ATP hydrolysis, and also support the chaperone activity of ClpB.     

梭热杆菌纤维小体研究进展

梭热杆菌(Clostridium thermocellum)是一种嗜热厌氧细菌,通过分泌大量纤维素酶高效降解纤维素.根据作用纤维素的不同部位,梭热杆菌分泌的纤维素酶分为内切纤维素酶和外切纤维素酶.纤维小体是由支架蛋白、锚定元件、黏合蛋白、纤维素结合域和催化单位组成的复合体,其独特的结构,使得它可以比真菌纤维素酶更紧密地结合到纤维素表面,这个复合结构结合着多种催化单位,而此特殊的结构是梭热杆菌高效降解纤维素的必要条件.近年来,为更深入透彻地了解纤维小体的结构与功能进行了大量的研究工作,现对相关研究进展进行综述,并给出了未来可能的发展方向.