Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase.

Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants. Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa. The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD). The cenD gene was cloned and sequenced. It encodes a polypeptide of 747 amino acids. Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da. Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C. fimi. Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases. The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III. CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration. The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB.     

Structural and functional relationships in two families of beta-1,4-glycanases.

CenA and Cex are beta-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C. fimi-type cellulose-binding domains. CenA and Cex belong to families B and F, respectively, in the classification of beta-1,4-glucanases and beta-1,4-xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 beta-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p-nitrophenyl-beta-D-xylobioside is 14,385 min-1.mM-1 at 25 degrees C; the corresponding kcat/Km for p-nitrophenyl-beta-D-cellobioside hydrolysis is 296 min-1.mM-1.     

Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi.

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonas fimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are greater than 60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulose-binding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the beta-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.     

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.     

Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.     

纤维素酶分子结构和功能研究进展

概述了近10年来利用结构生物学和蛋白质工程技术在纤维素酶分子结构和功能方面研究的进展,包括：酶分子结构域的拆分、催化域和纤维素结合结构域的结构和功能的研究,纤维素酶的分子折叠.并展望了该领域的研究前景.     

Structural and functional analysis of a bacterial cellulase by proteolysis

CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases.     

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3453 nucleotides and encodes a protein of 1151 amino acids with a molecular mass of 126408 Da. The deduced amino acid sequence of CBPA contained one domain highly similar to a catalytic domain of glycosyl hydrolases belonging to family 9, two linker-like domains and four domains of unknown function. Among the four domains of unknown function, the domains 1 and 2 region had significant homology in amino acid sequence with the cellulose-binding domains in the family 9 glycosyl hydrolases. The cloned gene was inserted into an expression vector, pBAD-TOPO, and expressed in Escherichia coli as a fused protein. The fused protein was detected by immunoblotting using antiserum against CBPA.     

Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996     

Bioinformatics analysis of bacteriophage and prophage endolysin domains

Endolysins as a class of antibacterial enzymes are expected to become a very useful tool for many purposes to control spreading of, e.g., multiresistant bacteria in different environments. Their antimicrobial properties could be broadened or altered by mutagenesis, domain swapping or gene shuffling. Therefore, the specific designing of endolysins to achieve their desired properties is challenging. This work is focused on the in silico analysis of protein domains presence in sequences of phage and prophage endolysins, followed by the study of variety of domain combinations in the individual endolysin types. The multiple sequence alignment of endolysin sequences revealed the recognition of sequence types with typical domain arrangement and conserved amino acids, divided according to the target substrate in bacterial cell walls. The five protein families of catalytic domains are specifically occurring in dependence of bacterial Gram-type. The presence, types and numbers of binding domains within endolysin sequences were also studied. The obtained results enable a more targeted design of endolysins with required antimicrobial properties.     

Characterization of genes fromThermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains

Two genes fromThermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (PglA). ThexynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The genepglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and PglA are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of PglA and AmyB fromT. thermosulfurigenes EM1.     

Characterization of genes fromThermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains

Two genes fromThermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (PglA). ThexynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The genepglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and PglA are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of PglA and AmyB fromT. thermosulfurigenes EM1.     

Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides.

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.     

Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes

The entire nucleotide sequence of the Bacillus brevis grsB gene encoding the gramicidin S synthetase 2, which activates and condenses the four amino acids proline, valine, ornithine and leucine has been determined. The gene contains an open reading frame of 13,359 bp which encodes a protein of 4453 amino acids with a predicted Mr of 510,287. The gene is located within the gramicidin S biosynthetic operon, also containing the genes grsT and grsA, whose nucleotide sequences have been determined previously. Within the GrsB amino acid sequence four conserved and repeated domains of about 600 amino acids (45-50% identity) have been identified. The four domains are separated by non-homologous sequences of about 500 amino acids. The domains also share a high degree of similarity (20-70%) with eight peptide synthetases of bacterial and fungal origin as well as with conserved sequences of nine other adenylate-forming enzymes of diverse origin. On the basis of sequence homology and functional similarities, we infer that those enzymes share a common evolutionary origin and present a phylogenetic tree for this superfamily of domain-bearing enzymes.     

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.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

Protease propeptide structures,mechanisms of activation,and functions

Abstract

Proteases are a diverse group of hydrolytic enzymes, ranging from single-domain catalytic molecules to sophisticated multi-functional macromolecules. Human proteases are divided into five mechanistic classes: aspartate, cysteine, metallo, serine and threonine proteases, based on the catalytic mechanism of hydrolysis. As a protective mechanism against uncontrolled proteolysis, proteases are often produced and secreted as inactive precursors, called zymogens, containing inhibitory N-terminal propeptides. Protease propeptide structures vary considerably in length, ranging from dipeptides and propeptides of about 10 amino acids to complex multifunctional prodomains with hundreds of residues. Interestingly, sequence analysis of the different protease domains has demonstrated that propeptide sequences present higher heterogeneity compared with their catalytic domains. Therefore, we suggest that protease inhibition targeting propeptides might be more specific and have less off-target effects than classical inhibitors. The roles of propeptides, besides keeping protease latency, include correct folding of proteases, compartmentalization, liganding, and functional modulation. Changes in the propeptide sequence, thus, have a tremendous impact on the cognate enzymes. Small modifications of the propeptide sequences modulate the activity of the enzymes, which may be useful as a therapeutic strategy. This review provides an overview of known human proteases, with a focus on the role of their propeptides. We review propeptide functions, activation mechanisms, and possible therapeutic applications.     

Active-site peptide "fingerprinting" of glycosidases in complex mixtures by mass spectrometry. Discovery of a novel retaining beta-1,4-glycanase in Cellulomonas fimi

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.