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PDBsum entry 1c5h

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Hydrolase PDB id
1c5h
Jmol
Contents
Protein chain
185 a.a. *
Waters ×146
* Residue conservation analysis
PDB id:
1c5h
Name: Hydrolase
Title: Hydrogen bonding and catalysis: an unexpected explanation for how a single amino acid substitution can change the ph optimum of a glycosidase
Structure: Endo-1,4-beta-xylanase. Chain: a. Fragment: catalytic domain. Synonym: bcx. Engineered: yes. Mutation: yes
Source: Bacillus circulans. Organism_taxid: 1397. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.
Resolution:
1.55Å     R-factor:   0.194    
Authors: M.D.Joshi,G.Sidhu,I.Pot,G.D.Brayer,S.G.Withers,L.P.Mcintosh
Key ref:
M.D.Joshi et al. (2000). Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J Mol Biol, 299, 255-279. PubMed id: 10860737 DOI: 10.1006/jmbi.2000.3722
Date:
24-Nov-99     Release date:   12-May-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P09850  (XYNA_BACCI) -  Endo-1,4-beta-xylanase
Seq:
Struc:
213 a.a.
185 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.8  - Endo-1,4-beta-xylanase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     metabolic process   4 terms 
  Biochemical function     hydrolase activity     4 terms  

 

 
DOI no: 10.1006/jmbi.2000.3722 J Mol Biol 299:255-279 (2000)
PubMed id: 10860737  
 
 
Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase.
M.D.Joshi, G.Sidhu, I.Pot, G.D.Brayer, S.G.Withers, L.P.McIntosh.
 
  ABSTRACT  
 
The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.
 
  Selected figure(s)  
 
Figure 6.
Figure 6. A stereo-illustration of the structural conformations of key active-site residues of the N35D BCX glycosyl-enzyme intermediate (N35D-2FXb) (dark gray) superimposed upon those of the WT glycosyl-enzyme intermediate (WT-2FXb) (light gray) (pH 7.5). Potential hydrogen bonds are indicated by broken yellow lines, oxygen atoms are shown in red and nitrogen atoms in blue. Modified Glu78-2FXb (Glu78*) is covalently attached to a 2-fluoroxylobiosyl (2FXb) moeity where the proximal saccharide is distorted to a ^2,5B conformation in both N35D-2FXb and WT-2FXb. A crystallographically identifiable water (Wat) molecule that is proposed to function in the deglycosylation step of the reaction is indicated by a red sphere. The most notable change is a reduction in the distance between Asn35 N^δ2/Asp35 O^δ2 and Glu172 from 3.3 Å in WT-2FXb to 2.7 Å in N35D-2FXb. See Table 3 for a listing of additional interatomic distances.
Figure 10.
Figure 10. The proposed double-displacement retaining mechanism of N35D BCX. In the glycosylation step, Asp35 and Glu172 function together in serving the role of the acid/base catalyst, whereas deprotonated Glu78 is the nucleophile. In the glycosyl-enzyme intermediate, Asp35-Glu172 interact strongly with coupled ionizations, pK[a1] 1.9-3.4 and pK[a2]>9. Due to this pK[a] cycling, they can now serve as a general base in the deglycosylation step of the reaction.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 299, 255-279) copyright 2000.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21287606 H.Webb, B.M.Tynan-Connolly, G.M.Lee, D.Farrell, F.O'Meara, C.R.Søndergaard, K.Teilum, C.Hewage, L.P.McIntosh, and J.E.Nielsen (2011).
Remeasuring HEWL pK(a) values by NMR spectroscopy: Methods, analysis, accuracy, and implications for theoretical pK(a) calculations.
  Proteins, 79, 685-702.  
20225927 A.Pollet, J.A.Delcour, and C.M.Courtin (2010).
Structural determinants of the substrate specificities of xylanases from different glycoside hydrolase families.
  Crit Rev Biotechnol, 30, 176-191.  
19769747 A.Pollet, S.Sansen, G.Raedschelders, K.Gebruers, A.Rabijns, J.A.Delcour, and C.M.Courtin (2009).
Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA.
  FEBS J, 276, 3916-3927.
PDB codes: 2b42 3hd8
19371088 M.L.Raber, S.O.Arnett, and C.A.Townsend (2009).
A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase.
  Biochemistry, 48, 4959-4971.  
19531602 T.Beliën, I.J.Joye, J.A.Delcour, and C.M.Courtin (2009).
Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability.
  Protein Eng Des Sel, 22, 587-596.  
19411257 T.J.Morley, L.M.Willis, C.Whitfield, W.W.Wakarchuk, and S.G.Withers (2009).
A new sialidase mechanism: bacteriophage K1F endo-sialidase is an inverting glycosidase.
  J Biol Chem, 284, 17404-17410.  
18498103 D.C.Bas, D.M.Rogers, and J.H.Jensen (2008).
Very fast prediction and rationalization of pKa values for protein-ligand complexes.
  Proteins, 73, 765-783.  
18384043 G.André-Leroux, J.G.Berrin, J.Georis, F.Arnaut, and N.Juge (2008).
Structure-based mutagenesis of Penicillium griseofulvum xylanase using computational design.
  Proteins, 72, 1298-1307.  
17189477 B.M.Tynan-Connolly, and J.E.Nielsen (2007).
Redesigning protein pKa values.
  Protein Sci, 16, 239-249.  
17513587 T.Beliën, S.Van Campenhout, M.Van Acker, J.Robben, C.M.Courtin, J.A.Delcour, and G.Volckaert (2007).
Mutational analysis of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum reveals comprehensive insights into their inhibitor insensitivity.
  Appl Environ Microbiol, 73, 4602-4608.  
17588227 Y.He, J.Xu, and X.M.Pan (2007).
A statistical approach to the prediction of pK(a) values in proteins.
  Proteins, 69, 75-82.  
16751556 T.Kim, E.J.Mullaney, J.M.Porres, K.R.Roneker, S.Crowe, S.Rice, T.Ko, A.H.Ullah, C.B.Daly, R.Welch, and X.G.Lei (2006).
Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive.
  Appl Environ Microbiol, 72, 4397-4403.  
15659364 F.De Lemos Esteves, T.Gouders, J.Lamotte-Brasseur, S.Rigali, and J.M.Frère (2005).
Improving the alkalophilic performances of the Xyl1 xylanase from Streptomyces sp. S38: structural comparison and mutational analysis.
  Protein Sci, 14, 292-302.  
16116276 H.Shibuya, S.Kaneko, and K.Hayashi (2005).
A single amino acid substitution enhances the catalytic activity of family 11 xylanase at alkaline pH.
  Biosci Biotechnol Biochem, 69, 1492-1497.  
15652973 T.Collins, C.Gerday, and G.Feller (2005).
Xylanases, xylanase families and extremophilic xylanases.
  FEMS Microbiol Rev, 29, 3.  
14638688 A.Hirata, M.Adachi, A.Sekine, Y.N.Kang, S.Utsumi, and B.Mikami (2004).
Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum.
  J Biol Chem, 279, 7287-7295.
PDB codes: 1q6c 1q6d 1q6e 1q6f 1q6g
15129441 A.Olivera-Nappa, B.A.Andrews, and J.A.Asenjo (2004).
A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity.
  Biotechnol Bioeng, 86, 573-586.  
14717693 B.Synstad, S.Gåseidnes, D.M.Van Aalten, G.Vriend, J.E.Nielsen, and V.G.Eijsink (2004).
Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase.
  Eur J Biochem, 271, 253-262.  
15096627 F.de Lemos Esteves, V.Ruelle, J.Lamotte-Brasseur, B.Quinting, and J.M.Frère (2004).
Acidophilic adaptation of family 11 endo-beta-1,4-xylanases: modeling and mutational analysis.
  Protein Sci, 13, 1209-1218.  
14660638 J.K.McCarthy, A.Uzelac, D.F.Davis, and D.E.Eveleigh (2004).
Improved catalytic efficiency and active site modification of 1,4-beta-D-glucan glucohydrolase A from Thermotoga neapolitana by directed evolution.
  J Biol Chem, 279, 11495-11502.  
15130470 M.F.Amaya, A.G.Watts, I.Damager, A.Wehenkel, T.Nguyen, A.Buschiazzo, G.Paris, A.C.Frasch, S.G.Withers, and P.M.Alzari (2004).
Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase.
  Structure, 12, 775-784.
PDB codes: 1s0i 1s0j 1s0k 2ah2
15451095 T.A.Tahir, A.Durand, K.Gebruers, A.Roussel, G.Williamson, and N.Juge (2004).
Functional importance of Asp37 from a family 11 xylanase in the binding to two proteinaceous xylanase inhibitors from wheat.
  FEMS Microbiol Lett, 239, 9.  
12657781 A.J.Oakley, T.Heinrich, C.A.Thompson, and M.C.Wilce (2003).
Characterization of a family 11 xylanase from Bacillus subtillis B230 used for paper bleaching.
  Acta Crystallogr D Biol Crystallogr, 59, 627-636.
PDB code: 1igo
12831897 D.Shallom, and Y.Shoham (2003).
Microbial hemicellulases.
  Curr Opin Microbiol, 6, 219-228.  
12761390 J.Le Nours, C.Ryttersgaard, L.Lo Leggio, P.R.Østergaard, T.V.Borchert, L.L.Christensen, and S.Larsen (2003).
Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum.
  Protein Sci, 12, 1195-1204.
PDB codes: 1hjq 1hjs 1hju
14567703 P.J.O'Brien, and T.Ellenberger (2003).
Human alkyladenine DNA glycosylase uses acid-base catalysis for selective excision of damaged purines.
  Biochemistry, 42, 12418-12429.  
14596624 S.S.Lee, S.Yu, and S.G.Withers (2003).
Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase.
  Biochemistry, 42, 13081-13090.  
11916711 A.Tomschy, R.Brugger, M.Lehmann, A.Svendsen, K.Vogel, D.Kostrewa, S.F.Lassen, D.Burger, A.Kronenberger, A.P.van Loon, L.Pasamontes, and M.Wyss (2002).
Engineering of phytase for improved activity at low pH.
  Appl Environ Microbiol, 68, 1907-1913.  
12413546 A.Vasella, G.J.Davies, and M.Böhm (2002).
Glycosidase mechanisms.
  Curr Opin Chem Biol, 6, 619-629.  
12146938 D.J.Vocadlo, J.Wicki, K.Rupitz, and S.G.Withers (2002).
Mechanism of Thermoanaerobacterium saccharolyticum beta-xylosidase: kinetic studies.
  Biochemistry, 41, 9727-9735.  
12146939 D.J.Vocadlo, J.Wicki, K.Rupitz, and S.G.Withers (2002).
A case for reverse protonation: identification of Glu160 as an acid/base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant.
  Biochemistry, 41, 9736-9746.  
  16233324 M.Nishimoto, M.Kitaoka, and K.Hayashi (2002).
Employing chimeric xylanases to identify regions of an alkaline xylanase participating in enzyme activity at basic pH.
  J Biosci Bioeng, 94, 395-400.  
11900558 T.Kaper, H.H.van Heusden, B.van Loo, A.Vasella, J.van der Oost, and W.M.de Vos (2002).
Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.
  Biochemistry, 41, 4147-4155.  
11738173 D.L.Zechel, and S.G.Withers (2001).
Dissection of nucleophilic and acid-base catalysis in glycosidases.
  Curr Opin Chem Biol, 5, 643-649.  
11717493 J.Wouters, J.Georis, D.Engher, J.Vandenhaute, J.Dusart, J.M.Frere, E.Depiereux, and P.Charlier (2001).
Crystallographic analysis of family 11 endo-beta-1,4-xylanase Xyl1 from Streptomyces sp. S38.
  Acta Crystallogr D Biol Crystallogr, 57, 1813-1819.
PDB code: 1hix
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.