PDBsum entry 1c5h

Hydrolase

PDB id: 1c5h

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

Protein chain

P09850  (XYNA_NIACI) -  Endo-1,4-beta-xylanase from Niallia circulans

Seq: 213 a.a.

Struc: 185 a.a.*

* 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.
• Reaction: Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.

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.