Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum.

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.

Noncatalytic docking domains of cellulosomes of anaerobic fungi.

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA from Piromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.

Do domain interactions of glycosyl hydrolases from Clostridium thermocellum contribute to protein thermostability?

Cellulolytic and hemicellulolytic enzymes usually have a domain composition. The mutual influence of a cellulose-binding domain and a catalytic domain was investigated with cellobiohydrolase CelK and xylanase XynZ from Clostridium thermocellum. CelK is composed of an N-terminal family IV cellulose-binding domain (CBDIV(CelK)), a family 9 glycosyl hydrolase domain (Gh9(CelK)) and a dockerin domain (DD). CelK without the DD, (CBDIV-Gh9)(CelK) and CBDIV(CelK) bound cellulose. The thermostability of (CBDIV-Gh9)(CelK) was significantly higher than that of CBDIV(CelK) and Gh9(CelK). The temperature optima of (CBDIV-Gh9)(CelK) and Gh9(CelK) were 65 and 45 degrees C, respectively. XynZ consists of an N-terminal feruloyl esterase domain (FAE(XynZ)), a linker (L), a family VI CBD (CBDVI(XynZ)), a DD and a xylanase domain. FAE(XynZ) and (FAE-L-CBDVI)(XynZ), used in the present study did not bind cellulose, but both were highly thermostable. Replacement of CBDVI(XynZ) with CBDIV(CelK) resulted in chimeras with feruloyl esterase activity and the ability to bind cellulose. CBDIV(CelK)-FAE(XynZ) bound cellulose with parameters similar to that of (CBDIV-Gh9)(CelK). (FAE-L)(XynZ)-CBDIV(CelK) and FAE(XynZ)-CBDIV(CelK) had lower relative affinities and binding capacities than those of (CBDIV-Gh9)(CelK). The three chimeras were much less thermostable than FAE(XynZ) and (FAE-L-CBDVI)(XynZ). The results indicate that domains of glycosyl hydrolases are not randomly combined and that domain interactions affect properties of these domain-structured enzymes.

Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin.

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.

Properties and mutation analysis of the CelK cellulose-binding domain from the Clostridium thermocellum cellulosome.

The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD(CelK)) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 micromol/g of cellulose and relative affinities (K(r)) of 2.33 and 9.87 liters/g, respectively. The CBD(CelK) is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD(CelK) also binds to the soluble polysaccharides lichenin, glucomannan, and barley beta-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD(CelK) contains 1 mol of calcium per mol. The CBD(CelK) has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD(CelK) four highly conserved aromatic residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N(CelK). The CBD-N(CelK) and D192A retained binding parameters close to that of the intact CBD(CelK), W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K(r) and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD(CelK) led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD(CelK) and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD(CelK) are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD(N1) Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD(CelK) correctly folded.

Crystallization and preliminary X-ray analysis of the Clostridium thermocellum cellulosome xylanase Z feruloyl esterase domain.

Feruloyl esterases cleave ferulic acid from arabinoxylan and pectin. Feruloyl groups are believed to crosslink the polysaccharide chain within the polymer and to link hemicellulose to lignin, which may play a role in controlling the growth of plants. The Clostridium thermocellum cellulosome xylanase Z feruloyl esterase was expressed in Escherichia coli, purified and crystallized. The crystals diffract to 2.4 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.14, b = 63.77, c = 79.57 A. Assuming one molecule per asymmetric unit, the Matthews coefficient is calculated to be 1.87 A(3) Da(-1), which corresponds to a solvent content of 34%.

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.

Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.

Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum: evidence for gene duplication and recombination.

The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of cbhA (V. V. Zverlov et al., J. Bacteriol. 180:3091-3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate that celK and cbhA in the genome of C. thermocellum have evolved through gene duplication and recombination of domain coding sequences. celK without a dockerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65 degrees C, respectively. It hydrolyzed p-nitrophenyl-beta-D-cellobioside with a Km and a Vmax of 1.67 microM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60 degrees C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.

Two genes of the anaerobic fungus Orpinomyces sp. strain PC-2 encoding cellulases with endoglucanase activities may have arisen by gene duplication.

A cDNA designated celE cloned from Orpinomyces PC-2 consisted of an open reading frame encoding a polypeptide (CelE) of 477 amino acids. CelE was highly homologous to CelBs of Orpinomyces (72.3% identity) and neocallimastix (67.9% identity) and like them it had a non-catalytic repeated peptide domain (NCRPD) at the C-terminal end. The catalytic domain of CelE was homologous to glycosyl hydrolases of Family 5, found in several anaerobic bacteria. The gene of celE was devoid of introns. The recombinant proteins CelE and CelB of Orpinomyces PC-2 randomly hydrolyzed carboxymethylcellulose and cello-oligosaccharides in the pattern of endoglucanases. The results indicated that a gene of bacterial origin was duplicated to form celE and celB of Orpinomyces PC-2.

Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex.

Two cDNAs encoding two cellulases, CelA and CelC, were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. Nucleotide sequencing revealed that the celA cDNA (1,558 bp) and celC cDNA (1,628 bp) had open reading frames encoding polypeptides of 459 (CelA) and 449 (CelC) amino acids, respectively. The two cDNAs were 76.9 and 67.7% identical at the nucleotide and amino acid levels, respectively. Analysis of the deduced amino acid sequences showed that starting from the N termini, both CelA and CelC had signal peptides, which were followed by noncatalytic repeated peptide domains (NCRPD) containing two repeated sequences of 33 to 40 amino acid residues functioning as docking domains. The NCRPDs and the catalytic domains were separated by linker sequences. The NCRPDs were homologous to those found in several hydrolases of anaerobic fungi, whereas the catalytic domains were homologous to the catalytic domains of fungal cellobiohydrolases and bacterial endoglucanases. The linker sequence of CelA contained predominantly glutamine and proline residues, while that of CelC contained mainly threonine residues. CelA and CelC did not have a typical cellulose binding domain (CBD). CelA and CelC expressed in E. coli rapidly decreased the viscosity of carboxymethyl cellulose (CMC), indicating that there was endoglucanase activity. In addition, they produced cellobiose from CMC, acid-swollen cellulose, and cellotetraose, suggesting that they had cellobiohydrolase activity. The optimal activity conditions with CMC as the substrate were pH 4.3 to 6.8 and 50 degrees C for CelA and pH 4.6 to 7.0 and 40 degrees C for CelC. Despite the lack of a CBD, CelC displayed a high affinity for microcrystalline cellulose, whereas CelA did not.

Sequencing of a 1,3-1,4-beta-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin.

A 971-bp cDNA, designated licA, was obtained from a library of Orpinomyces sp. strain PC-2 constructed in Escherichia coli. It had an open reading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanase; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molecular mass of 27,929 Da. The deduced amino acid sequence had high homology with bacterial beta-glucanases, particularly in the central regions and toward the C-terminal halves of bacterial enzymes. LicA had no homology with plant beta-glucanases. The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-glucanase produced in E. coli cells was found in the culture medium and periplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino acid residues of the signal peptide were removed when the enzyme was produced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium. It had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The Km and Vmax values with lichenin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 micromol/min/mg, respectively. With barley beta-glucan as the substrate, the corresponding values were 0.91 mg/ml and 5,320 micromol/min/mg. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pustulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose. LicA represented the first 1,3-1,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.

Composition and primary structure of the F1F0 ATP synthase from the obligately anaerobic bacterium Clostridium thermoaceticum.

The subunit composition and primary structure of the proton-translocating F1F0 ATP synthase have been determined in Clostridium thermoaceticum. The isolated enzyme has a subunit composition identical to that of the F1F0 ATP synthase purified from Clostridium thermoautotrophicum (A. Das, D. M. Ivey, and L. G. Ljungdahl, J. Bacteriol. 179:1714-1720, 1997), both having six different polypeptides. The molecular masses of the six subunits were 60, 50, 32, 17, 19, and 8 kDa, and they were identified as alpha, beta, gamma, delta, epsilon, and c, respectively, based on their reactivity with antibodies against the F1 ATPase purified from C. thermoautotrophicum and by comparing their N-terminal amino acid sequences with that deduced from the cloned genes of the C. thermoaceticum atp operon. The subunits a and b found in many bacterial ATP synthases could not be detected either in the purified ATP synthase or crude membranes of C. thermoaceticum. The C. thermoaceticum atp operon contained nine genes arranged in the order atpI (i), atpB (a), atpE (c), atpF (b), atpH (delta), atpA (alpha), atpG (gamma), atpD (beta), and atpC (epsilon). The deduced protein sequences of the C. thermoaceticum ATP synthase subunits were comparable with those of the corresponding subunits from Escherichia coli, thermophilic Bacillus strain PS3, Rhodospirillum rubrum, spinach chloroplasts, and the cyanobacterium Synechococcus strain PCC 6716. The analysis of total RNA by Northern hybridization experiments reveals the presence of transcripts (mRNA) of the genes i, a, and b subunits not found in the isolated enzyme. Analysis of the nucleotide sequence of the atp genes reveals overlap of the structural genes for the i and a subunits and the presence of secondary structures (in the b gene) which could influence the posttranscriptional regulation of the corresponding genes.

Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum.

The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium. The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E. coli.

Monocentric and polycentric anaerobic fungi produce structurally related cellulases and xylanases.

Cellulase and xylanase cDNAs were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. The cellulase cDNA (celB) was 1.8 kb long with an open reading frame (ORF) coding for a polypeptide of 471 amino acids, and the xylanase cDNA (xynA) was 1.2 kb long with an ORF encoding a polypeptide of 362 amino acids. Single transcripts of 1.9 kb for celB and 1.5 kb for xynA were detected in total RNA of Orpinomyces grown on Avicel. Genomic DNA regions coding for CelA and XynA were devoid of introns. The enzymes were highly homologous (80 to 85% identity) to the corresponding enzymes of the monocentric anaerobic fungus Neocallimastix patriciarum and, like those, contained in addition to a catalytic domain, a noncatalytic repeated peptide domain (NCRPD). The Orpinomyces xylanase contained one catalytic domain and thus differed from the Neocallimastix xylanase, which had two similar catalytic domains (H. J. Gilbert, G. P. Hazlewood, J. I. Lauie, C. G. Orpin, and G. P. Xue, Mol. Microbiol. 6:2065-2072, 1992). Two peptides corresponding to the catalytic domain and the NCRPD of XynA were synthesized, and antibodies against them were raised and affinity column purified. The antibodies against the catalytic domain peptide reacted specifically with the xylanases of Orpinomyces and Neocallimastix, while the antibodies against the NCRPD reacted with many (at least eight) extracellular proteins of Orpinomyces and Neocallimastix, suggesting that the NCRPD is present in a number of polypeptides.

Dissociation of the cellulosome of Clostridium thermocellum in the presence of ethylenediaminetetraacetic acid occurs with the formation of trucated polypeptides.

The cellulosome of Clostridium thermocellum JW20 consists of 14-26 different polypeptides as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intact cellulosomes hydrolyzes crystalline cellulose in the presence of Ca2+ and thiols. This activity is inhibited by ethylenediaminetetraacetic acid (EDTA). Ca is incorporated into the cellulosome and tightly bound as demonstrated using 45Ca added to the growth medium. Upon incubation in 50 mM Tris (pH 7.5), 0.1 M NaCl, and 5 mM EDTA at 37 degrees C, Ca is released from the cellulosome, which disintegrates into polypeptides. The SDS-PAGE pattern of cellulosomal polypeptides is remarkably different after the EDTA treatment when compared to this pattern of untreated cellulosomes. Polypeptide bands corresponding to molecular masses of 160, 98, 76, 91, and 54 kDa disappear, and new bands of masses 150, 132, 91, 71, 57, and 46 kDa appear. N-terminal analyses of the 98, 76, 91, and 71 kDa polypeptides show that the 91 and 71 kDa polypeptides are truncated products of the 98 and 76 kDa polypeptides, respectively. The 76 and 71 kDa polypeptides correspond to CelS [Wang, W. K., Kruus K., & Wu, J. H. D. (1993) J. Bacteriol. 175, 1293-1302]. The 71 kDa polypeptide is formed from the 76 kDa polypeptide during the EDTA treatment, by a cleavage that occurs at asparagine residue 681. It involves the removal of 60 amino acid residues from the C-terminal end. All catalytic subunits so far characterized contain an asparagine residue corresponding to residue 681 of CelS. This residue is part of the conserved duplicated region found in catalytically active subunits, and it is postulated that several of these subunits also are truncated by the EDTA treatment. The polypeptides truncated by the EDTA treatment reduced Ca binding capacities compared to their native subunits, indicating a Ca-binding site within the conserved duplicated region. This region has been implicated in the binding of the catalytic peptides to the scaffolding polypeptide CipA, which is a structural protein of the cellulosome and has a strong affinity for calcium.

Structural role of calcium for the organization of the cellulosome of Clostridium thermocellum.

The cellulosome of Clostridium thermocellum is a multipolypeptide complex of structural and catalytic subunits. Several of the catalytic subunits have at the carboxyl end a conserved duplicated region (CDR) which interacts with internally repeated elements (IREs) of scaffolding subunits such as CipA. This interaction requires calcium. The two parts of the CDR region here designated CDR1 and CDR2 (closest to the carboxyl end) each consist of about 20 amino acids residues. As shown in our previous paper [Choi, S.K., & Ljungdahl, L.G. (1996) Biochemistry 35, 4897-4905], treatment of the cellulosome with ethylenediaminetetraacetic acid (EDTA) under aerobic conditions disintegrates the cellulosome with formation of truncated catalytic subunits. The cleavage is at a specific asparagine residue located within CDR1 and occurs with complete loss of CDR2. Two branched peptides containing the amino acid sequences of CDR1 and CDR2 (designated bCDR1 and bCDR2) were synthesized, and specific antibodies were raised against them. These antibodies did not cross react with bCDR1 or bCDR2, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, it was observed that about 15 subunits of the cellulosome reacted with anti-bCDR1 and anti-bCDR2. In a similar experiment with EDTA-treated cellulosomes, these subunits reacted with anti-bCDR1 but not with anti-bCDR2, showing that they lost the bCDR2 epitope and were truncated. The peptide bCDR1 binds calcium, whereas bCDR2 does not. Furthermore, bCDR1 but not bCDR2 binds to CipA, presumably at IRE regions. This binding requires calcium. A model is proposed for the binding of the catalytic subunits to CipA which involves CDR1, an IRE, and calcium.

Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae.

A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L. Li and L. G. Ljungdahl, Appl. Environ. Microbiol. 60:3160-3166, 1994). Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium. S. cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction. Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4. These proteins were recognized by anti-APX-II antibody. The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S. cerevisiae. Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media. No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region. A comparison of the A. pullulans and S. cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S. cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S. cerevisiae cells.

A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 is highly homologous to vertebrate cyclophilin B.

A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.

Biomass degrading enzymes from anaerobic rumen fungi.

Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous mammals. They produce a complete set of polysaccharide hydrolytic enzymes which efficiently degrade plant cell-walls. This article summarizes the present work on biomass degrading enzymes with special emphasis on their cellulases, xylanases, and esterases from both monocentric and polycentric anaerobic fungi. The possibility of the cellulosome or xylanosome-like high molecular mass complexes in the fungi are also discussed.