SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2164037667> ?p ?o ?g. }

Showing items 1 to 100 of ±109 with 100 items per page.

W2164037667 endingPage "17530" @default.
W2164037667 startingPage "17523" @default.
W2164037667 abstract "Crystal structure analysis of Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) indicated that the enzyme contained a single calcium binding site that did not exhibit structural features typical of the EF-hand motif. Isothermal titration calorimetry revealed that XYLA binds calcium with a K a of 4.9 × 104m−1 and a stoichiometry consistent with one calcium binding site per molecule of enzyme. Occupancy of the calcium binding domain with its ligand protected XYLA from proteinase and thermal inactivation and increased the melting temperature of the enzyme from 60.8 to 66.5 °C. However, the addition of calcium or EDTA did not influence the catalytic activity of the xylanase. Replacement of the calcium binding domain, which is located within loop 7 of XYLA, with the corresponding short loop from Cex (aCellulomonas fimi xylanase/exoglucanase), did not significantly alter the biochemical properties of the enzyme. These data suggest that the primary function of the calcium binding domain is to increase the stability of the enzyme against thermal unfolding and proteolytic attack. To understand further the nature of the calcium binding domain of XYLA, four variants of the xylanase, D256A, N261A, D262A, and XYLA′′′, in which Asp-256, Asn-261, and Asp-262 had all been changed to alanine, were constructed. These mutated enzymes did not show any significant binding to Ca2+, indicating that Asp-256, Asn-261, and Asp-262 play a pivotal role in the affinity of XYLA for the divalent cation. In the presence or absence of calcium, XYLA′′′ exhibited thermal stability similar to that of the native enzyme bound to Ca2+ ions, although the variant was sensitive to proteinase inactivation. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed. Crystal structure analysis of Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) indicated that the enzyme contained a single calcium binding site that did not exhibit structural features typical of the EF-hand motif. Isothermal titration calorimetry revealed that XYLA binds calcium with a K a of 4.9 × 104m−1 and a stoichiometry consistent with one calcium binding site per molecule of enzyme. Occupancy of the calcium binding domain with its ligand protected XYLA from proteinase and thermal inactivation and increased the melting temperature of the enzyme from 60.8 to 66.5 °C. However, the addition of calcium or EDTA did not influence the catalytic activity of the xylanase. Replacement of the calcium binding domain, which is located within loop 7 of XYLA, with the corresponding short loop from Cex (aCellulomonas fimi xylanase/exoglucanase), did not significantly alter the biochemical properties of the enzyme. These data suggest that the primary function of the calcium binding domain is to increase the stability of the enzyme against thermal unfolding and proteolytic attack. To understand further the nature of the calcium binding domain of XYLA, four variants of the xylanase, D256A, N261A, D262A, and XYLA′′′, in which Asp-256, Asn-261, and Asp-262 had all been changed to alanine, were constructed. These mutated enzymes did not show any significant binding to Ca2+, indicating that Asp-256, Asn-261, and Asp-262 play a pivotal role in the affinity of XYLA for the divalent cation. In the presence or absence of calcium, XYLA′′′ exhibited thermal stability similar to that of the native enzyme bound to Ca2+ ions, although the variant was sensitive to proteinase inactivation. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed. Endo-β1,4-xylanases (xylanases; EC 3.2.1.8) catalyze the cleavage of internal β-1,4 glycosidic linkages in the backbone of xylans (1Biely P. Trends Biotechnol. 1985; 3: 286-290Abstract Full Text PDF Scopus (798) Google Scholar, 2Gilbert H.J. Hazlewood G.P. J. Gen. Microbiol. 1993; 139: 187-194Crossref Scopus (313) Google Scholar). Using hydrophobic cluster analysis, the catalytic domains of glycosyl hydrolases have been classified into 57 different enzyme families (3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1177) Google Scholar). Members of each family are thought to have evolved from a common ancestral sequence. Xylanases belong to either Family 10 or 11 (4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1756) Google Scholar). Recently, hydrophobic cluster analysis has shown that several enzyme families have common folds, suggesting that they evolved from a single sequence (5Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.-P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7090-7094Crossref PubMed Scopus (511) Google Scholar). Support for the concept of a common evolutionary link among some glycosyl hydrolase families is provided by the conservation of the three-dimensional structure of enzymes from different families, particularly in the vicinity of the active site, and the observation that glycosidases belonging to a specific clan cleave glycosidic bonds by the same mechanism (5Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.-P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7090-7094Crossref PubMed Scopus (511) Google Scholar, 6Jenkins J. Lo Leggio L. Harris G. Pickersgill R. FEBS Lett. 1995; 362: 281-285Crossref PubMed Scopus (236) Google Scholar).In general, glycosyl hydrolases are unusually resistant to proteolytic attack and thermal inactivation (7Fontes C.M.G. Hall J. Hirst B.H. Hazlewood G.P. Gilbert H.J. Appl. Microbiol. Biotechnol. 1995; 43: 52-57Crossref PubMed Scopus (73) Google Scholar). Although calcium ions often play an important role in conferring structural stability on proteins, only two cellulases, a hybrid Bacillus glucanase and CelD fromClostridium thermocellum, have been shown to bind to Ca2+ and to both Ca2+ and Zn2+ions, respectively (8Keitel T. Simon O. Borriss R. Heinmann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5287-5291Crossref PubMed Scopus (194) Google Scholar, 9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). High concentrations of calcium enhanced the thermostability of both enzymes and decreased the K m of CelD for 4-nitrophenyl-β-cellobioside (9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Welfle K. Misselwitz R. Welfle H. Politz O. Borriss R. Eur. J. Biochem. 1995; 229: 726-735Crossref PubMed Google Scholar).Recently, the three-dimensional structures of the catalytic domain of four Family 10 enzymes have been solved (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 12White A. Withers S.G. Gilkes N.R. Rose D.R. Biochemistry. 1994; 33: 12546-12552Crossref PubMed Scopus (163) Google Scholar, 13Derewenda U. Swenson L. Green R. Wei Y. Morosoli R. Shareck F. Kluepfel D. Derewenda Z. J. Biol. Chem. 1994; 269: 20811-20814Abstract Full Text PDF PubMed Google Scholar, 14Dominguez R. Souchon H. Spinelli S. Dauter Z. Wilson K. Chauvaux S. Beguin P. Alzari P.M. Nat. Struct. Biol. 1995; 2: 569-576Crossref PubMed Scopus (169) Google Scholar). They all consist of an (α/β)8-fold barrel structure in which two conserved glutamates function as the catalytic nucleophile and acid/base catalytic residues, respectively. Xylanase A (XYLA) 1The abbreviations used are: XYLA, xylanase A; MUC, 4-methylumbelliferyl-β-cellobioside; PAGE, polyacrylamide gel electrophoresis; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; MOPS, 4-morpholinepropanesulfonic acid; T m, thermal denaturation temperature. 1The abbreviations used are: XYLA, xylanase A; MUC, 4-methylumbelliferyl-β-cellobioside; PAGE, polyacrylamide gel electrophoresis; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; MOPS, 4-morpholinepropanesulfonic acid; T m, thermal denaturation temperature. from Pseudomonas fluorescens subsp. cellulosa is a modular enzyme comprising an NH2-terminal cellulose binding domain linked to a COOH-terminal catalytic domain. The catalytic domain is unique within Family 10 enzymes as it is the only xylanase described to date which contains a calcium binding site (located in loop 7) (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and it is the only xylanase from either mesophilic or thermophilic microorganisms which has been shown to be sensitive to proteinases (15Hall J. Ali S. Surani M.A. Hazlewood G.P. Clark A.J. Simmons J.P. Hirst B.H. Gilbert H.J. Bio/Technology. 1993; 11: 376-379Crossref PubMed Scopus (7) Google Scholar). The function(s) of the calcium binding domain in XYLA and the structural basis for the enzyme's sensitivity to proteinases remain to be elucidated.The objective of this report is to determine the role of the calcium binding domain in XYLA. The data presented show that occupation of the calcium binding loop with its ligand protected the enzyme from thermal inactivation, thermal unfolding, and proteolytic attack. A mutant of XYLA in which the key residues of the calcium binding domain were replaced by alanine exhibited thermal stability similar to that of XYLA complexed with Ca2+ ions; however, the xylanase variant was susceptible to cleavage by chymotrypsin. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed.DISCUSSIONThe data presented in this paper showed that theK a of XYLA for calcium is 4.9 × 104m−1, which represents a relatively weak affinity compared with EF-hand calcium binding domains that bind their ligands with K a values of 105–109m−1 (29Seamon K.B. Kretsinger R.H. Spiro T.G. Calcium in Biology. John Wiley and Sons, New York1983: 3-51Google Scholar). The low affinity of XYLA for the divalent ion could reflect the environment of P. fluorescens subsp. cellulosa. The bacterium was isolated from soil samples of neutral pH in which Ca2+would be at a concentration >1 mm, sufficient to saturate XYLA (30Russell E.W. Wild A. Soil Conditions and Plant Growth. 11th Ed. Longman, London1988: 151Google Scholar). Inspection of the location of the calcium binding domain in the xylanase suggested that its structure may affect the catalytic properties of the enzyme as Tyr-255, which apparently forms part of subsite B, is positioned on the Ca2+ binding loop (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). However, the activity of the enzyme was not significantly influenced by either the disruption of the metal binding domain, through amino acid substitution or EDTA treatment, or saturation of the domain with its appropriate ligand. This suggests that either Tyr-255 does not play a pivotal role in xylose binding at site B, or the change in conformation of loop 7 through calcium binding does not influence the ability of the tyrosine residue to participate in substrate binding. It is also apparent that the conformational changes associated with calcium binding to loop 7, which influenced the biophysical properties of the enzyme, did not affect the structure of the extended substrate binding cleft.The binding of the calcium binding loop to its ligand caused a substantial effect on the biophysical properties of XYLA. The enzyme was less sensitive to thermal inactivation, which reflected a general increase in the stability of the protein, as evidenced by a considerable increase in the T m of XYLA in the presence of the divalent cation. These data are in agreement with several previous studies demonstrating that calcium stabilizes the structure of many proteins (9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Welfle K. Misselwitz R. Welfle H. Politz O. Borriss R. Eur. J. Biochem. 1995; 229: 726-735Crossref PubMed Google Scholar). Substituting Asp-256 or Asn-261 for alanine resulted in a substantial destabilization of the enzyme, whereas the D262A mutation resulted in a modest reduction in the thermal stability of the enzyme. It could be argued that these data suggest that Asp-262 is the key residue involved in calcium binding; however, ligand binding assays clearly showed that replacing Asp-256, Asn-261, or Asp-262 with alanine resulted in no detectable affinity of the XYLA variants for the divalent cation. Interestingly, XYLA′′′, in which Asp-256, Asn-261, and Asp-262 had all been replaced with alanine, exhibited thermal stability similar to that of native XYLA bound to calcium. This could reflect the location of Asp-256, Asn-261, and Asp-262 in loop 7. The electronegative carbonyl group of Asn-261 and the negatively charged carboxylic groups of Asp-256 and Asp-262 would repulse each other, resulting in a destabilization of the loop. Either the binding of calcium to these amino acids or their replacement with alanine would prevent the repulsive effects from occurring, resulting in a stabilization of the loop and thus the whole protein. This view is consistent with the acid-pair hypothesis proposed by Reid and Hodges (31Reid R.E. Hodges R.S. J. Theor. Biol. 1980; 84: 401-444Crossref PubMed Scopus (85) Google Scholar) which states that the Ca2+ affinity of EF-hand domains reflects an electrostatic compromise in which the attraction of divalent cations for anionic ligands, such as carboxylate groups, is counterbalanced by the interligand repulsion between coordinating oxygens. As D262A was more stable than D256A and N261A, it is likely that the major repulsive effects occur between Asp-262 and either Asp-256 or Asn-261.Data presented in this report demonstrate that the initial target for proteinase attack of XYLA is within the calcium binding domain in loop 7. It is likely, therefore, that the binding of calcium to this region tightens the conformation of the loop, making it less susceptible to enzymic cleavage. The transient nature of the 29-kDa peptide released by chymotrypsin suggests that hydrolysis of peptide bonds in the calcium binding loop caused a significant destabilization of the structure of XYLA, resulting in the very rapid subsequent hydrolysis of the molecule by the proteinase. In contrast to XYLA, XYLA′′′ was susceptible to proteinase attack in the presence and absence of calcium. These data suggest that the calcium binding loop in XYLA′′′ is more flexible than in the native enzyme (when complexed with its ligand), making it more accessible to proteinase cleavage.From the discussion above, it is apparent that the major role of the calcium binding domain in XYLA is to stabilize the extended structure of loop 7, either against thermal unfolding or proteinase attack. As Ca2+ only protects XYLA from thermal inactivation at temperatures above 55 °C, and the natural habitat ofPseudomonas is normally at a temperature lower than 30 °C, it is unlikely that the enhanced thermal stability afforded by calcium plays an important role in the survival of XYLA. We propose that the major function of the calcium binding domain of the xylanase is to protect the enzyme from proteinase attack. This view is supported by the unusual stability of other xylanases, from both mesophilic and thermophilic microorganisms, to proteolytic attack (7Fontes C.M.G. Hall J. Hirst B.H. Hazlewood G.P. Gilbert H.J. Appl. Microbiol. Biotechnol. 1995; 43: 52-57Crossref PubMed Scopus (73) Google Scholar), suggesting that this property exerts a strong selection pressure on the evolution of xylanases.To date only 5 of the 28 known Family 10 xylanases contain an extended loop 7. Although inspection of the extended loop in XYLA,Prevotella ruminicola XYLA (XYNA PRERU), Bacteroides ovatus XYLA (XYNA BACOV), and Bacillus stearothermophilus XYLN1 and XYN2 (XYN1 BACST and XYN2 BACST, respectively) did not reveal motifs, such as the EF-hand, which are characteristic of calcium binding domains, it is apparent that loop 7 in XYLA did bind to the divalent metal ion. As XYN1 and 2 BACST function at elevated temperatures, and XYNA PRERU and XYNA BACOV in environments that contain high levels of proteinase activity, it is likely that loop 7 in these xylanases is stabilized, possibly though interactions with metal ions or other sequences within the respective proteins. The central unanswered questions are why do only 5 Family 10 xylanases contain an extended loop 7, and how did the calcium binding domain in XYLA evolve? Loop 7 may confer specific catalytic properties on the enzyme. However, the observation that the biochemical characteristics of XYLA are not altered when the extended loop is replaced with the corresponding shorter loop from Cex argues against this view. It is possible that the ancestral protein that gave rise to Family 10 xylanases contained an extended loop 7 that destabilized the protein, and through natural selection deletions within the loop have resulted in the evolution of stable xylanases. Mutations in loop 7 of the ancestral sequence, which gave rise to XYLA, generated a calcium binding domain that stabilized the loop, hence there was no requirement for the loop to be reduced in size in the Pseudomonasenzyme. An alternative possibility is that the extended loop 7, in 5 of the 28 Family 10 xylanases, is the result of a DNA insertion into a DNA sequence that gave rise to the genes encoding the 5 enzymes. InxynA the inserted DNA subsequently acquired mutations such that it encoded a calcium binding domain in XYLA. To explore this hypothesis further, the primary structure of Family 10 xylanases were subject to phylogenetic analysis using parsimony methods. Initially, all sequence positions (410) were included in the analysis, which suggested a relationship between the xylanases containing the extended loop 7 (data not shown). To remove any bias due to the inclusion of extended loop 7 in the alignments, the corresponding sequence positions were removed and the analysis repeated (Fig. 10). It is interesting that the relationship between 4 of the 5 xylanases is maintained, indicating that the respective genes have evolved from a common ancestral sequence that contained a DNA insertion in the region encoding loop 7. However, this conclusion must be viewed with some caution as it is apparent that there has been considerable horizontal gene transfer between Family 10 xylanase genes (evidenced by the fact that very similar sequences occur in taxonomically diverse groups of organisms), and the bootstrap scores suggest that some clades are not particularly stable. Based on the relationship between thePseudomonas xylanase (XYNA PSEFL) and the clade containing the actinomycete (GUX CELFI and XYNA STRLI) and fungal (XYNA PENCH, GUNF FUSOX, and XYNA PENCH) xylanases, one might hypothesize that the latter had evolved, following loss of the extended loop 7, from XYNA PSEFL. However, additional sequence information from this region of the tree is needed to validate this hypothesis.To conclude, data presented in this report clearly showed that the extended loop 7 of Pseudomonas XYLA was stabilized by a calcium binding loop. Whether the corresponding extended loops of XYNA PRERU, XYNA BACOV, XYN1 BACST, and XYN2 BACST are also stabilized by binding to divalent metal ions, remains to be elucidated. Endo-β1,4-xylanases (xylanases; EC 3.2.1.8) catalyze the cleavage of internal β-1,4 glycosidic linkages in the backbone of xylans (1Biely P. Trends Biotechnol. 1985; 3: 286-290Abstract Full Text PDF Scopus (798) Google Scholar, 2Gilbert H.J. Hazlewood G.P. J. Gen. Microbiol. 1993; 139: 187-194Crossref Scopus (313) Google Scholar). Using hydrophobic cluster analysis, the catalytic domains of glycosyl hydrolases have been classified into 57 different enzyme families (3Henrissat B. Bairoch A. Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1177) Google Scholar). Members of each family are thought to have evolved from a common ancestral sequence. Xylanases belong to either Family 10 or 11 (4Henrissat B. Bairoch A. Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1756) Google Scholar). Recently, hydrophobic cluster analysis has shown that several enzyme families have common folds, suggesting that they evolved from a single sequence (5Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.-P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7090-7094Crossref PubMed Scopus (511) Google Scholar). Support for the concept of a common evolutionary link among some glycosyl hydrolase families is provided by the conservation of the three-dimensional structure of enzymes from different families, particularly in the vicinity of the active site, and the observation that glycosidases belonging to a specific clan cleave glycosidic bonds by the same mechanism (5Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.-P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7090-7094Crossref PubMed Scopus (511) Google Scholar, 6Jenkins J. Lo Leggio L. Harris G. Pickersgill R. FEBS Lett. 1995; 362: 281-285Crossref PubMed Scopus (236) Google Scholar). In general, glycosyl hydrolases are unusually resistant to proteolytic attack and thermal inactivation (7Fontes C.M.G. Hall J. Hirst B.H. Hazlewood G.P. Gilbert H.J. Appl. Microbiol. Biotechnol. 1995; 43: 52-57Crossref PubMed Scopus (73) Google Scholar). Although calcium ions often play an important role in conferring structural stability on proteins, only two cellulases, a hybrid Bacillus glucanase and CelD fromClostridium thermocellum, have been shown to bind to Ca2+ and to both Ca2+ and Zn2+ions, respectively (8Keitel T. Simon O. Borriss R. Heinmann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5287-5291Crossref PubMed Scopus (194) Google Scholar, 9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). High concentrations of calcium enhanced the thermostability of both enzymes and decreased the K m of CelD for 4-nitrophenyl-β-cellobioside (9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Welfle K. Misselwitz R. Welfle H. Politz O. Borriss R. Eur. J. Biochem. 1995; 229: 726-735Crossref PubMed Google Scholar). Recently, the three-dimensional structures of the catalytic domain of four Family 10 enzymes have been solved (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 12White A. Withers S.G. Gilkes N.R. Rose D.R. Biochemistry. 1994; 33: 12546-12552Crossref PubMed Scopus (163) Google Scholar, 13Derewenda U. Swenson L. Green R. Wei Y. Morosoli R. Shareck F. Kluepfel D. Derewenda Z. J. Biol. Chem. 1994; 269: 20811-20814Abstract Full Text PDF PubMed Google Scholar, 14Dominguez R. Souchon H. Spinelli S. Dauter Z. Wilson K. Chauvaux S. Beguin P. Alzari P.M. Nat. Struct. Biol. 1995; 2: 569-576Crossref PubMed Scopus (169) Google Scholar). They all consist of an (α/β)8-fold barrel structure in which two conserved glutamates function as the catalytic nucleophile and acid/base catalytic residues, respectively. Xylanase A (XYLA) 1The abbreviations used are: XYLA, xylanase A; MUC, 4-methylumbelliferyl-β-cellobioside; PAGE, polyacrylamide gel electrophoresis; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; MOPS, 4-morpholinepropanesulfonic acid; T m, thermal denaturation temperature. 1The abbreviations used are: XYLA, xylanase A; MUC, 4-methylumbelliferyl-β-cellobioside; PAGE, polyacrylamide gel electrophoresis; ITC, isothermal titration calorimetry; DSC, differential scanning calorimetry; MOPS, 4-morpholinepropanesulfonic acid; T m, thermal denaturation temperature. from Pseudomonas fluorescens subsp. cellulosa is a modular enzyme comprising an NH2-terminal cellulose binding domain linked to a COOH-terminal catalytic domain. The catalytic domain is unique within Family 10 enzymes as it is the only xylanase described to date which contains a calcium binding site (located in loop 7) (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and it is the only xylanase from either mesophilic or thermophilic microorganisms which has been shown to be sensitive to proteinases (15Hall J. Ali S. Surani M.A. Hazlewood G.P. Clark A.J. Simmons J.P. Hirst B.H. Gilbert H.J. Bio/Technology. 1993; 11: 376-379Crossref PubMed Scopus (7) Google Scholar). The function(s) of the calcium binding domain in XYLA and the structural basis for the enzyme's sensitivity to proteinases remain to be elucidated. The objective of this report is to determine the role of the calcium binding domain in XYLA. The data presented show that occupation of the calcium binding loop with its ligand protected the enzyme from thermal inactivation, thermal unfolding, and proteolytic attack. A mutant of XYLA in which the key residues of the calcium binding domain were replaced by alanine exhibited thermal stability similar to that of XYLA complexed with Ca2+ ions; however, the xylanase variant was susceptible to cleavage by chymotrypsin. The role of the calcium binding domain in vivo and the possible mechanism by which the domain evolved are discussed. DISCUSSIONThe data presented in this paper showed that theK a of XYLA for calcium is 4.9 × 104m−1, which represents a relatively weak affinity compared with EF-hand calcium binding domains that bind their ligands with K a values of 105–109m−1 (29Seamon K.B. Kretsinger R.H. Spiro T.G. Calcium in Biology. John Wiley and Sons, New York1983: 3-51Google Scholar). The low affinity of XYLA for the divalent ion could reflect the environment of P. fluorescens subsp. cellulosa. The bacterium was isolated from soil samples of neutral pH in which Ca2+would be at a concentration >1 mm, sufficient to saturate XYLA (30Russell E.W. Wild A. Soil Conditions and Plant Growth. 11th Ed. Longman, London1988: 151Google Scholar). Inspection of the location of the calcium binding domain in the xylanase suggested that its structure may affect the catalytic properties of the enzyme as Tyr-255, which apparently forms part of subsite B, is positioned on the Ca2+ binding loop (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). However, the activity of the enzyme was not significantly influenced by either the disruption of the metal binding domain, through amino acid substitution or EDTA treatment, or saturation of the domain with its appropriate ligand. This suggests that either Tyr-255 does not play a pivotal role in xylose binding at site B, or the change in conformation of loop 7 through calcium binding does not influence the ability of the tyrosine residue to participate in substrate binding. It is also apparent that the conformational changes associated with calcium binding to loop 7, which influenced the biophysical properties of the enzyme, did not affect the structure of the extended substrate binding cleft.The binding of the calcium binding loop to its ligand caused a substantial effect on the biophysical properties of XYLA. The enzyme was less sensitive to thermal inactivation, which reflected a general increase in the stability of the protein, as evidenced by a considerable increase in the T m of XYLA in the presence of the divalent cation. These data are in agreement with several previous studies demonstrating that calcium stabilizes the structure of many proteins (9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Welfle K. Misselwitz R. Welfle H. Politz O. Borriss R. Eur. J. Biochem. 1995; 229: 726-735Crossref PubMed Google Scholar). Substituting Asp-256 or Asn-261 for alanine resulted in a substantial destabilization of the enzyme, whereas the D262A mutation resulted in a modest reduction in the thermal stability of the enzyme. It could be argued that these data suggest that Asp-262 is the key residue involved in calcium binding; however, ligand binding assays clearly showed that replacing Asp-256, Asn-261, or Asp-262 with alanine resulted in no detectable affinity of the XYLA variants for the divalent cation. Interestingly, XYLA′′′, in which Asp-256, Asn-261, and Asp-262 had all been replaced with alanine, exhibited thermal stability similar to that of native XYLA bound to calcium. This could reflect the location of Asp-256, Asn-261, and Asp-262 in loop 7. The electronegative carbonyl group of Asn-261 and the negatively charged carboxylic groups of Asp-256 and Asp-262 would repulse each other, resulting in a destabilization of the loop. Either the binding of calcium to these amino acids or their replacement with alanine would prevent the repulsive effects from occurring, resulting in a stabilization of the loop and thus the whole protein. This view is consistent with the acid-pair hypothesis proposed by Reid and Hodges (31Reid R.E. Hodges R.S. J. Theor. Biol. 1980; 84: 401-444Crossref PubMed Scopus (85) Google Scholar) which states that the Ca2+ affinity of EF-hand domains reflects an electrostatic compromise in which the attraction of divalent cations for anionic ligands, such as carboxylate groups, is counterbalanced by the interligand repulsion between coordinating oxygens. As D262A was more stable than D256A and N261A, it is likely that the major repulsive effects occur between Asp-262 and either Asp-256 or Asn-261.Data presented in this report demonstrate that the initial target for proteinase attack of XYLA is within the calcium binding domain in loop 7. It is likely, therefore, that the binding of calcium to this region tightens the conformation of the loop, making it less susceptible to enzymic cleavage. The transient nature of the 29-kDa peptide released by chymotrypsin suggests that hydrolysis of peptide bonds in the calcium binding loop caused a significant destabilization of the structure of XYLA, resulting in the very rapid subsequent hydrolysis of the molecule by the proteinase. In contrast to XYLA, XYLA′′′ was susceptible to proteinase attack in the presence and absence of calcium. These data suggest that the calcium binding loop in XYLA′′′ is more flexible than in the native enzyme (when complexed with its ligand), making it more accessible to proteinase cleavage.From the discussion above, it is apparent that the major role of the calcium binding domain in XYLA is to stabilize the extended structure of loop 7, either against thermal unfolding or proteinase attack. As Ca2+ only protects XYLA from thermal inactivation at temperatures above 55 °C, and the natural habitat ofPseudomonas is normally at a temperature lower than 30 °C, it is unlikely that the enhanced thermal stability afforded by calcium plays an important role in the survival of XYLA. We propose that the major function of the calcium binding domain of the xylanase is to protect the enzyme from proteinase attack. This view is supported by the unusual stability of other xylanases, from both mesophilic and thermophilic microorganisms, to proteolytic attack (7Fontes C.M.G. Hall J. Hirst B.H. Hazlewood G.P. Gilbert H.J. Appl. Microbiol. Biotechnol. 1995; 43: 52-57Crossref PubMed Scopus (73) Google Scholar), suggesting that this property exerts a strong selection pressure on the evolution of xylanases.To date only 5 of the 28 known Family 10 xylanases contain an extended loop 7. Although inspection of the extended loop in XYLA,Prevotella ruminicola XYLA (XYNA PRERU), Bacteroides ovatus XYLA (XYNA BACOV), and Bacillus stearothermophilus XYLN1 and XYN2 (XYN1 BACST and XYN2 BACST, respectively) did not reveal motifs, such as the EF-hand, which are characteristic of calcium binding domains, it is apparent that loop 7 in XYLA did bind to the divalent metal ion. As XYN1 and 2 BACST function at elevated temperatures, and XYNA PRERU and XYNA BACOV in environments that contain high levels of proteinase activity, it is likely that loop 7 in these xylanases is stabilized, possibly though interactions with metal ions or other sequences within the respective proteins. The central unanswered questions are why do only 5 Family 10 xylanases contain an extended loop 7, and how did the calcium binding domain in XYLA evolve? Loop 7 may confer specific catalytic properties on the enzyme. However, the observation that the biochemical characteristics of XYLA are not altered when the extended loop is replaced with the corresponding shorter loop from Cex argues against this view. It is possible that the ancestral protein that gave rise to Family 10 xylanases contained an extended loop 7 that destabilized the protein, and through natural selection deletions within the loop have resulted in the evolution of stable xylanases. Mutations in loop 7 of the ancestral sequence, which gave rise to XYLA, generated a calcium binding domain that stabilized the loop, hence there was no requirement for the loop to be reduced in size in the Pseudomonasenzyme. An alternative possibility is that the extended loop 7, in 5 of the 28 Family 10 xylanases, is the result of a DNA insertion into a DNA sequence that gave rise to the genes encoding the 5 enzymes. InxynA the inserted DNA subsequently acquired mutations such that it encoded a calcium binding domain in XYLA. To explore this hypothesis further, the primary structure of Family 10 xylanases were subject to phylogenetic analysis using parsimony methods. Initially, all sequence positions (410) were included in the analysis, which suggested a relationship between the xylanases containing the extended loop 7 (data not shown). To remove any bias due to the inclusion of extended loop 7 in the alignments, the corresponding sequence positions were removed and the analysis repeated (Fig. 10). It is interesting that the relationship between 4 of the 5 xylanases is maintained, indicating that the respective genes have evolved from a common ancestral sequence that contained a DNA insertion in the region encoding loop 7. However, this conclusion must be viewed with some caution as it is apparent that there has been considerable horizontal gene transfer between Family 10 xylanase genes (evidenced by the fact that very similar sequences occur in taxonomically diverse groups of organisms), and the bootstrap scores suggest that some clades are not particularly stable. Based on the relationship between thePseudomonas xylanase (XYNA PSEFL) and the clade containing the actinomycete (GUX CELFI and XYNA STRLI) and fungal (XYNA PENCH, GUNF FUSOX, and XYNA PENCH) xylanases, one might hypothesize that the latter had evolved, following loss of the extended loop 7, from XYNA PSEFL. However, additional sequence information from this region of the tree is needed to validate this hypothesis.To conclude, data presented in this report clearly showed that the extended loop 7 of Pseudomonas XYLA was stabilized by a calcium binding loop. Whether the corresponding extended loops of XYNA PRERU, XYNA BACOV, XYN1 BACST, and XYN2 BACST are also stabilized by binding to divalent metal ions, remains to be elucidated. The data presented in this paper showed that theK a of XYLA for calcium is 4.9 × 104m−1, which represents a relatively weak affinity compared with EF-hand calcium binding domains that bind their ligands with K a values of 105–109m−1 (29Seamon K.B. Kretsinger R.H. Spiro T.G. Calcium in Biology. John Wiley and Sons, New York1983: 3-51Google Scholar). The low affinity of XYLA for the divalent ion could reflect the environment of P. fluorescens subsp. cellulosa. The bacterium was isolated from soil samples of neutral pH in which Ca2+would be at a concentration >1 mm, sufficient to saturate XYLA (30Russell E.W. Wild A. Soil Conditions and Plant Growth. 11th Ed. Longman, London1988: 151Google Scholar). Inspection of the location of the calcium binding domain in the xylanase suggested that its structure may affect the catalytic properties of the enzyme as Tyr-255, which apparently forms part of subsite B, is positioned on the Ca2+ binding loop (11Harris G.W. Jenkins J.A. Connerton I. Cummings N. Lo Leggio L. Scott M. Hazlewood G.P. Laurie J.I. Gilbert H.J. Pickersgill R.W. Structure. 1994; 2: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). However, the activity of the enzyme was not significantly influenced by either the disruption of the metal binding domain, through amino acid substitution or EDTA treatment, or saturation of the domain with its appropriate ligand. This suggests that either Tyr-255 does not play a pivotal role in xylose binding at site B, or the change in conformation of loop 7 through calcium binding does not influence the ability of the tyrosine residue to participate in substrate binding. It is also apparent that the conformational changes associated with calcium binding to loop 7, which influenced the biophysical properties of the enzyme, did not affect the structure of the extended substrate binding cleft. The binding of the calcium binding loop to its ligand caused a substantial effect on the biophysical properties of XYLA. The enzyme was less sensitive to thermal inactivation, which reflected a general increase in the stability of the protein, as evidenced by a considerable increase in the T m of XYLA in the presence of the divalent cation. These data are in agreement with several previous studies demonstrating that calcium stabilizes the structure of many proteins (9Chauvaux S. Souchon H. Alzari P.M. Chariot P. Beguin P. J. Biol. Chem. 1995; 270: 9757-9762Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Welfle K. Misselwitz R. Welfle H. Politz O. Borriss R. Eur. J. Biochem. 1995; 229: 726-735Crossref PubMed Google Scholar). Substituting Asp-256 or Asn-261 for alanine resulted in a substantial destabilization of the enzyme, whereas the D262A mutation resulted in a modest reduction in the thermal stability of the enzyme. It could be argued that these data suggest that Asp-262 is the key residue involved in calcium binding; however, ligand binding assays clearly showed that replacing Asp-256, Asn-261, or Asp-262 with alanine resulted in no detectable affinity of the XYLA variants for the divalent cation. Interestingly, XYLA′′′, in which Asp-256, Asn-261, and Asp-262 had all been replaced with alanine, exhibited thermal stability similar to that of native XYLA bound to calcium. This could reflect the location of Asp-256, Asn-261, and Asp-262 in loop 7. The electronegative carbonyl group of Asn-261 and the negatively charged carboxylic groups of Asp-256 and Asp-262 would repulse each other, resulting in a destabilization of the loop. Either the binding of calcium to these amino acids or their replacement with alanine would prevent the repulsive effects from occurring, resulting in a stabilization of the loop and thus the whole protein. This view is consistent with the acid-pair hypothesis proposed by Reid and Hodges (31Reid R.E. Hodges R.S. J. Theor. Biol. 1980; 84: 401-444Crossref PubMed Scopus (85) Google Scholar) which states that the Ca2+ affinity of EF-hand domains reflects an electrostatic compromise in which the attraction of divalent cations for anionic ligands, such as carboxylate groups, is counterbalanced by the interligand repulsion between coordinating oxygens. As D262A was more stable than D256A and N261A, it is likely that the major repulsive effects occur between Asp-262 and either Asp-256 or Asn-261. Data presented in this report demonstrate that the initial target for proteinase attack of XYLA is within the calcium binding domain in loop 7. It is likely, therefore, that the binding of calcium to this region tightens the conformation of the loop, making it less susceptible to enzymic cleavage. The transient nature of the 29-kDa peptide released by chymotrypsin suggests that hydrolysis of peptide bonds in the calcium binding loop caused a significant destabilization of the structure of XYLA, resulting in the very rapid subsequent hydrolysis of the molecule by the proteinase. In contrast to XYLA, XYLA′′′ was susceptible to proteinase attack in the presence and absence of calcium. These data suggest that the calcium binding loop in XYLA′′′ is more flexible than in the native enzyme (when complexed with its ligand), making it more accessible to proteinase cleavage. From the discussion above, it is apparent that the major role of the calcium binding domain in XYLA is to stabilize the extended structure of loop 7, either against thermal unfolding or proteinase attack. As Ca2+ only protects XYLA from thermal inactivation at temperatures above 55 °C, and the natural habitat ofPseudomonas is normally at a temperature lower than 30 °C, it is unlikely that the enhanced thermal stability afforded by calcium plays an important role in the survival of XYLA. We propose that the major function of the calcium binding domain of the xylanase is to protect the enzyme from proteinase attack. This view is supported by the unusual stability of other xylanases, from both mesophilic and thermophilic microorganisms, to proteolytic attack (7Fontes C.M.G. Hall J. Hirst B.H. Hazlewood G.P. Gilbert H.J. Appl. Microbiol. Biotechnol. 1995; 43: 52-57Crossref PubMed Scopus (73) Google Scholar), suggesting that this property exerts a strong selection pressure on the evolution of xylanases. To date only 5 of the 28 known Family 10 xylanases contain an extended loop 7. Although inspection of the extended loop in XYLA,Prevotella ruminicola XYLA (XYNA PRERU), Bacteroides ovatus XYLA (XYNA BACOV), and Bacillus stearothermophilus XYLN1 and XYN2 (XYN1 BACST and XYN2 BACST, respectively) did not reveal motifs, such as the EF-hand, which are characteristic of calcium binding domains, it is apparent that loop 7 in XYLA did bind to the divalent metal ion. As XYN1 and 2 BACST function at elevated temperatures, and XYNA PRERU and XYNA BACOV in environments that contain high levels of proteinase activity, it is likely that loop 7 in these xylanases is stabilized, possibly though interactions with metal ions or other sequences within the respective proteins. The central unanswered questions are why do only 5 Family 10 xylanases contain an extended loop 7, and how did the calcium binding domain in XYLA evolve? Loop 7 may confer specific catalytic properties on the enzyme. However, the observation that the biochemical characteristics of XYLA are not altered when the extended loop is replaced with the corresponding shorter loop from Cex argues against this view. It is possible that the ancestral protein that gave rise to Family 10 xylanases contained an extended loop 7 that destabilized the protein, and through natural selection deletions within the loop have resulted in the evolution of stable xylanases. Mutations in loop 7 of the ancestral sequence, which gave rise to XYLA, generated a calcium binding domain that stabilized the loop, hence there was no requirement for the loop to be reduced in size in the Pseudomonasenzyme. An alternative possibility is that the extended loop 7, in 5 of the 28 Family 10 xylanases, is the result of a DNA insertion into a DNA sequence that gave rise to the genes encoding the 5 enzymes. InxynA the inserted DNA subsequently acquired mutations such that it encoded a calcium binding domain in XYLA. To explore this hypothesis further, the primary structure of Family 10 xylanases were subject to phylogenetic analysis using parsimony methods. Initially, all sequence positions (410) were included in the analysis, which suggested a relationship between the xylanases containing the extended loop 7 (data not shown). To remove any bias due to the inclusion of extended loop 7 in the alignments, the corresponding sequence positions were removed and the analysis repeated (Fig. 10). It is interesting that the relationship between 4 of the 5 xylanases is maintained, indicating that the respective genes have evolved from a common ancestral sequence that contained a DNA insertion in the region encoding loop 7. However, this conclusion must be viewed with some caution as it is apparent that there has been considerable horizontal gene transfer between Family 10 xylanase genes (evidenced by the fact that very similar sequences occur in taxonomically diverse groups of organisms), and the bootstrap scores suggest that some clades are not particularly stable. Based on the relationship between thePseudomonas xylanase (XYNA PSEFL) and the clade containing the actinomycete (GUX CELFI and XYNA STRLI) and fungal (XYNA PENCH, GUNF FUSOX, and XYNA PENCH) xylanases, one might hypothesize that the latter had evolved, following loss of the extended loop 7, from XYNA PSEFL. However, additional sequence information from this region of the tree is needed to validate this hypothesis. To conclude, data presented in this report clearly showed that the extended loop 7 of Pseudomonas XYLA was stabilized by a calcium binding loop. Whether the corresponding extended loops of XYNA PRERU, XYNA BACOV, XYN1 BACST, and XYN2 BACST are also stabilized by binding to divalent metal ions, remains to be elucidated. We thank Dr. Ian Fleet (UMIST) for doing the mass spectrometry and Judy Laurie for doing the NH2-terminal sequencing." @default.
W2164037667 created "2016-06-24" @default.
W2164037667 creator A5003461542 @default.
W2164037667 creator A5004810388 @default.
W2164037667 creator A5017782573 @default.
W2164037667 creator A5020696923 @default.
W2164037667 creator A5030104820 @default.
W2164037667 creator A5045334542 @default.
W2164037667 creator A5059560909 @default.
W2164037667 creator A5074033457 @default.
W2164037667 date "1997-07-01" @default.
W2164037667 modified "2023-09-28" @default.
W2164037667 title "Calcium Protects a Mesophilic Xylanase from Proteinase Inactivation and Thermal Unfolding" @default.
W2164037667 cites W1525347874 @default.
W2164037667 cites W1605341148 @default.
W2164037667 cites W1772341111 @default.
W2164037667 cites W1792723487 @default.
W2164037667 cites W1830745998 @default.
W2164037667 cites W1978230195 @default.
W2164037667 cites W1983967005 @default.
W2164037667 cites W1989089713 @default.
W2164037667 cites W2000500498 @default.
W2164037667 cites W2007807529 @default.
W2164037667 cites W2009766495 @default.
W2164037667 cites W2013260039 @default.
W2164037667 cites W2020357919 @default.
W2164037667 cites W2022510326 @default.
W2164037667 cites W2030085152 @default.
W2164037667 cites W2034453583 @default.
W2164037667 cites W2079150307 @default.
W2164037667 cites W2091418652 @default.
W2164037667 cites W2100837269 @default.
W2164037667 cites W2106882534 @default.
W2164037667 cites W2108218166 @default.
W2164037667 cites W2129728418 @default.
W2164037667 cites W2140807560 @default.
W2164037667 cites W2170264257 @default.
W2164037667 cites W2239384431 @default.
W2164037667 cites W4253833576 @default.
W2164037667 doi "https://doi.org/10.1074/jbc.272.28.17523" @default.
W2164037667 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9211898" @default.
W2164037667 hasPublicationYear "1997" @default.
W2164037667 type Work @default.
W2164037667 sameAs 2164037667 @default.
W2164037667 citedByCount "45" @default.
W2164037667 countsByYear W21640376672012 @default.
W2164037667 countsByYear W21640376672013 @default.
W2164037667 countsByYear W21640376672014 @default.
W2164037667 countsByYear W21640376672015 @default.
W2164037667 countsByYear W21640376672016 @default.
W2164037667 countsByYear W21640376672018 @default.
W2164037667 countsByYear W21640376672019 @default.
W2164037667 countsByYear W21640376672021 @default.
W2164037667 countsByYear W21640376672022 @default.
W2164037667 crossrefType "journal-article" @default.
W2164037667 hasAuthorship W2164037667A5003461542 @default.
W2164037667 hasAuthorship W2164037667A5004810388 @default.
W2164037667 hasAuthorship W2164037667A5017782573 @default.
W2164037667 hasAuthorship W2164037667A5020696923 @default.
W2164037667 hasAuthorship W2164037667A5030104820 @default.
W2164037667 hasAuthorship W2164037667A5045334542 @default.
W2164037667 hasAuthorship W2164037667A5059560909 @default.
W2164037667 hasAuthorship W2164037667A5074033457 @default.
W2164037667 hasBestOaLocation W21640376671 @default.
W2164037667 hasConcept C12554922 @default.
W2164037667 hasConcept C178790620 @default.
W2164037667 hasConcept C181199279 @default.
W2164037667 hasConcept C185592680 @default.
W2164037667 hasConcept C2775859485 @default.
W2164037667 hasConcept C519063684 @default.
W2164037667 hasConcept C523546767 @default.
W2164037667 hasConcept C54355233 @default.
W2164037667 hasConcept C55493867 @default.
W2164037667 hasConcept C63223902 @default.
W2164037667 hasConcept C86803240 @default.
W2164037667 hasConcept C95444343 @default.
W2164037667 hasConceptScore W2164037667C12554922 @default.
W2164037667 hasConceptScore W2164037667C178790620 @default.
W2164037667 hasConceptScore W2164037667C181199279 @default.
W2164037667 hasConceptScore W2164037667C185592680 @default.
W2164037667 hasConceptScore W2164037667C2775859485 @default.
W2164037667 hasConceptScore W2164037667C519063684 @default.
W2164037667 hasConceptScore W2164037667C523546767 @default.
W2164037667 hasConceptScore W2164037667C54355233 @default.
W2164037667 hasConceptScore W2164037667C55493867 @default.
W2164037667 hasConceptScore W2164037667C63223902 @default.
W2164037667 hasConceptScore W2164037667C86803240 @default.
W2164037667 hasConceptScore W2164037667C95444343 @default.
W2164037667 hasIssue "28" @default.
W2164037667 hasLocation W21640376671 @default.
W2164037667 hasOpenAccess W2164037667 @default.
W2164037667 hasPrimaryLocation W21640376671 @default.
W2164037667 hasRelatedWork W2061855233 @default.
W2164037667 hasRelatedWork W2078678552 @default.
W2164037667 hasRelatedWork W2114351110 @default.
W2164037667 hasRelatedWork W2138270807 @default.
W2164037667 hasRelatedWork W2290453688 @default.
W2164037667 hasRelatedWork W2315149664 @default.