Biomedical subjects

D Kluepfel

Publications and source records attributed to D Kluepfel.

At least 19 recordsLinked to original sources

PubMed2000

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A.

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

Glycoside Hydrolases↗

PubMed2000

Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase.

A thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XlnA). The screening system was tested using mutants randomized at position 49 of the S. lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1-blue bacteria. The resulting clones were screened for increased thermostability with respect to wild-type XlnA. Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants. The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000-20,000 clones.

Journal Article↗

PubMed1999

Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans.

The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA.

Amino Acid Sequence↗

PubMed1998

Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains.

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

Amino Acid Sequence↗

PubMed1998

Site-directed mutagenesis study of a conserved residue in family 10 glycanases: histidine 86 of xylanase A from Streptomyces lividans.

Xylanases from family 10 glycanases contain three conserved histidine residues in their active site. The role of H86 in the structure-function of xylanase A from Streptomyces lividans (XlnA) was studied by site-directed mutagenesis. Six mutant proteins (H86A/E/F/K/Q/W) were produced, purified and characterized. The six mutations reduced the affinity of XlnA towards xylan without having any major effect on the catalytic constant. All these mutations also lowered the pKa of the acid-base catalyst by 0.46-1.94 pH units. The mutations decreased the enzyme stability at 60 degrees C by up to 95% and the transition temperature by 2.2-5.8 degrees C. Unfolding of the protein with guanidine hydrochloride (GdnxHCl) showed that five out of six mutations decreased the concentration required to denature 50% of the XlnA, confirming the importance of H86 for the stability of the enzyme. The increase in m value ¿m=d(deltaG)/d[GdnxHCl]¿ also suggested the involvement of residue H86 in the structure of the denatured state of XlnA. It can be concluded from this study that this active site residue was conserved in family 10 glycanases for its function in maintaining the elevated pKa of the acid-base catalyst and in the stability of the protein, while being of little importance for the activity.

Binding Sites↗

PubMed1997

Endo-beta-1,4-xylanase families: differences in catalytic properties.

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 (formerly F) and 11 (formerly G) differ in their action on 4-O-methyl-D-glucurono-D-xylan and rhodymenan, a beta-1,3-beta-1,4-xylan. Two high molecular mass EXs (family 10), the Cryptococcus albidus EX and XlnA of Streptomyces lividans, liberate from glucuronoxylan aldotetrauronic acid as the shortest acidic fragment, and from rhodymenan an isomeric xylotriose of the structure Xyl beta 1-3Xyl beta 1-4Xyl as the shortest fragment containing a beta-1,3-linkage. Low molecular mass EXs (family 11), such as the Trichoderma reesei enzymes and XlnB and XlnC of S. lividans, liberate from glucuronoxylan an aldopentauronic acid as the shortest fragment, and from rhodymenan an isomeric xylotetraose as the shortest fragment containing a beta-1,3-linkage. The structure of the oligosaccharides was established by: NMR spectroscopy, mass spectrometry of per-O-methylated compounds and enzymic hydrolysis by beta-xylosidase and EX, followed by analysis of products by chromatography. The structures of the fragments define in the polysaccharides the linkages attacked and non-attacked by the enzymes. EXs of family 10 require a lower number of unsubstituted consecutive beta-1,4-xylopyranosyl units in the main chain and a lower number of consecutive beta-1,4-xylopyranosyl linkages in rhodymenan than EXs of family 11. These results, together with a greater catalytic versatility of EXs of family 10, suggest that EXs of family 10 have substrate binding sites smaller than those of EXs of family 11. This suggestion is in agreement with the finding that EXs of family 10 show higher affinity for shorter linear beta-1,4-xylooligosaccharides than EXs of family 11. The results are discussed with relevant literature data to understand better the structure-function relationship in this group of glycanases.

Endo-1,4-beta Xylanases↗

PubMed1997

Characterization of two important histidine residues in the active site of xylanase A from Streptomyces lividans, a family 10 glycanase.

The active site of xylanase A (XlnA) from Streptomyces lividans contains three histidine residues, two of which (H81 and H207) are almost completely conserved in family 10 glycanases. The structural analysis of the enzyme shows that H81 and H207 are part of an important hydrogen bond network in the vicinity of the two catalytic residues (E128 and E236). In order to investigate the role of these two histidine residues for the structure/function of XlnA, three mutant enzymes were produced at each position, namely, H81R/S/Y and H207E/K/R. The specific activity of these mutant enzymes is reduced by more than 95%, revealing the importance of these two residues for the catalytic function of XlnA. The kinetic parameters of the three more active enzymes were determined, of which mutation H207K increased the K(M) 3-fold. The k(cat) of the mutant enzymes is reduced proportionally to the specific activity. Furthermore, the pKa values of the two catalytic residues are decreased in all six mutations, demonstrating a role for H81 and H207 in the hydrogen bond network responsible for maintaining the ionization state of the two catalytic residues. In most cases, the unfolding of mutated XlnA in guanidine hydrochloride (Gdn-HCl) showed that the concentration required to denature 50% of the XlnA decreased, thus demonstrating the importance of those two residues for the stability of the enzyme. Moreover, the m value [m = d(deltaG)/d[Gdn-HCl]] for the unfolding of XlnA in Gdn-HCl is increased for each of the six mutations, suggesting that the mutant proteins have less residual structure in the denatured state than does the wild-type enzyme.

Amino Acid Sequence↗

PubMed1997

New alpha-L-arabinofuranosidase produced by Streptomyces lividans: cloning and DNA sequence of the abfB gene and characterization of the enzyme.

A fully secreted alpha-l-arabinofuranosidase was cloned from the homologous expression system of Streptomyces lividans. The gene, located upstream adjacent to the previously described xylanase A gene, was sequenced. It is divergently transcribed from the xlnA gene and the two genes are separated by an intercistronic region of 391nt which contains a palindromic AT-rich sequence. The deduced amino acid sequence of the protein shows that the enzyme contains a distinct catalytic domain which is linked to a specific xylan-binding domain by a linker region. The purified enzyme has a specific arabinofuranose-debranching activity on xylan from Gramineae, acts synergistically with the S. lividans xylanases and binds specifically to xylan. From small arabinoxylo-oligosides, it liberates arabinose and, after prolonged incubation, the purified enzyme exhibits some xylanolytic activity as well.

Amino Acid Sequence↗

PubMed1997

Protein secretion in streptomycetes.

Some aspects of the current knowledge on protein secretion in streptomycetes are presented including recent data on the identification of genes in the general secretory pathway, on the importance of the signal peptide structure and on the number of ribosome-binding sites inside signal peptides which can influence the production level of a gene product.

Amino Acid Sequence↗

PubMed1997

Asparagine-127 of xylanase A from Streptomyces lividans, a key residue in glycosyl hydrolases of superfamily 4/7: kinetic evidence for its involvement in stabilization of the catalytic intermediate.

Site-directed mutagenesis of asparagine-127 (N127) of xylanase A (XlnA) from Streptomyces lividans, belonging to family 10 and superfamily 4/7 of glycosyl hydrolases, was chosen to study the role of this conserved residue. The isosteric mutation N127D introduced did not affect the fold of XlnA as revealed by circular dichroism. Comparison of the kinetic constants of N127D and wild-type XlnA revealed a 70-fold decrease in the specificity constant (kcat/K(M)) towards birchwood xylan, which is attributed solely to the difference in the kcat value and indicates a role of N127 in stabilization of the catalytic intermediate. N127 also plays a role in maintaining the ionization states of the two catalytic residues, as shown by the modified pH profile of XlnA-N127D. Characterization of XlnA-N127D and the analysis of the three-dimensional structure of XlnA converge towards a stabilization role for N127 in the catalytic site of XlnA.

Asparagine↗

PubMed1996

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

Acetylation↗

PubMed1996

Purification and characterization of an acetyl xylan esterase produced by Streptomyces lividans.

The acetyl xylan esterase cloned homologously from Streptomyces lividans [Shareck, Biely, Morosoli and Kluepfel (1995) Gene 153, 105-109] was purified from culture filtrate of the overproducing strain S. lividans IAF43. The secreted enzyme had a molecular mass of 34 kDa and a pI of 9.0. Under the assay conditions with chemically acetylated birchwood xylan the kinetic constants of the enzyme were: specific activity, 715 units/mg, Km 7.94 mg/ml and Vmax 1977 units/mg. Optimal enzyme activity was obtained at 70 degrees C and pH 7.5. Hydrolysis assays with different acetylated substrates showed that the enzyme is specific for deacetylating the O-acetyl group of polysaccharides and is devoid of N-deacetylation activity. Sequential hydrolysis shows that its action is essential for the complete degradation of acetylated xylan by the xylanases of S. lividans.

Acetylesterase↗

PubMed1996

Cloning and sequencing of the secY homolog from Streptomyces lividans 1326.

Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces lividans (Sl). The nucleotide sequence of part of a 3.8-kb fragment showed that the secY homolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene. The deduced gene product of secY would have 437 amino acids (aa) and an M(r) of 47,200. Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively. Promoterprobe analyses indicated that the secY gene probably contains its own promoter.

Amino Acid Sequence↗

PubMed1996

Cloning of a secA homolog from Streptomyces lividans 1326 and overexpression in both S. lividans and Escherichia coli.

We cloned a gene encoding a SecA homolog from Streptomyces lividans 1326, a Gram-positive bacterium known to produce large amounts of extracellular proteins. A protein sequence alignment with the other bacterial SecA homologs revealed that S. lividans SecA shares from 39.5 to 44% identity with them, while it shares 34.2 to 37.2% identity with SecA homologs from plastids of algae and plants. We overexpressed the secA gene in S. lividans 1326 and Escherichia coli MM52 and in both cases we observed the production of a protein with an apparent molecular mass of 117.4 kDa. Although S. lividans SecA is similar to E. coli SecA, it does not complement a thermosensitive mutation in the E. coli secA gene. However, a hybrid polypeptide consisting of the N-terminal portion (first 242 amino acids) of the S. lividans SecA and the C-terminal portion (657 a.a.) of the wild-type E. coli SecA was able to complement this mutant.

Adenosine Triphosphatases↗

PubMed1996

Evidence for lysozyme-type mechanism of hydrolysis in xylanases.

In the last year several new xylanase three-dimensional structures were solved. Examination of these new structures in combination with recently obtained data from site-directed mutagenesis and kinetic analysis provided insights into the catalytic mechanism of xylanases. It is now possible to determine the type of mechanism by which xylanases hydrolyse a complex substrate such as xylan.

Amino Acid Sequence↗

PubMed1996

Expression and secretion of beta-glucuronidase and Pertussis toxin S1 by Streptomyces lividans.

Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coli beta-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the beta-glucuronidase gene was fused with the leader sequence produced up to 30 mg beta-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/1. The disappearance of the B. pertussis toxin S1 and beta-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.

Bordetella pertussis↗

PubMed1996

Increased xylanase yield in Streptomyces lividans: dependence on number of ribosome-binding sites.

The Streptomyces lividans xylanase A (XlnA) signal peptide (sp) was replaced with the signal peptides of either mannanase A (ManA) or cellulase A (CelA), two enzymes secreted by S. lividans. Depending on the location of the ribosome binding sites (RBS) with respect to a potential initiation codon, the length of the putative sps of ManA and CelA is either 34 or 43 amino acids and 27 or 46 amino acids, respectively. The sequence encoding these sps were fused to the xylanase A gene (xlnA). Clones harboring the short sps of ManA and CelA produced as much xylanase as the clone with the control wild-type sp sequence of XlnA. In clones containing the long sps of ManA and CelA, the XlnA production was enhanced 1.5- and 2.5-fold, respectively. These XlnA yields are reduced by half and one third respectively when the internal initiation codons of the long sp sequences of ManA and CelA are mutated. Since these clones exhibited the same transcription levels, the results indicate that both RBSs are used concomitantly in S. lividans to increase the enzyme production at the translational level. However, when the short and long sps of ManA were fused to the long CelA sp sequence, giving constructs containing respectively 3 and 4 RBSs, a decrease in xylanase production was observed.

Amino Acid Sequence↗

PubMed1996

Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans.

Starting from its translation initiation site, the Streptomyces lividans xylanase A signal peptide consists of 41 amino acids. This signal peptide was deleted and successively replaced with one of six signal peptides from other enzymes secreted by S. lividans and by a signal peptide from the outer membrane protein (LamB) of Escherichia coli. Deletion of the xylanase A signal peptide or modification of its cleavage site abolished secretion of the enzyme. Replacement with the signal peptides of either xylanase B, cellulase A, mannanase, or acetylxylan esterase produced equivalent amounts of xylanase A, while the signal peptides of cellulase B, xylanase C, and LamB secreted less enzyme than did the wild type. All the clones exhibited the same transcription levels, which indicated that the variations in xylanase production were due to the natures of the signal sequences.

Amino Acid Sequence↗