PDBsum entry: 1kr0

Hydrolase

PDB id: 1kr0

Contents

Protein chain

273 a.a. *

Ligands

NAG-NAG-NAG-NAG

SO4

Waters ×142

* Residue conservation analysis

PDB id:

1kr0

Name:

Hydrolase

Title:

Hevamine mutant d125a/y183f in complex with tetra-NAG

Structure:

Hevamine a. Chain: a. Engineered: yes. Mutation: yes

Source:

Hevea brasiliensis. Organism_taxid: 3981. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.92Å R-factor: 0.169 R-free: 0.204

Authors:

H.J.Rozeboom,B.W.Dijkstra

Key ref:

E.Bokma et al. (2002). Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis. Eur J Biochem, 269, 893-901. PubMed id: 11846790 DOI: 10.1046/j.0014-2956.2001.02721.x

Date:

08-Jan-02 Release date: 23-Jan-02

Protein chain

P23472  (CHLY_HEVBR) -  Hevamine-A

Seq: 311 a.a.

Struc: 273 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.3.2.1.14 - Chitinase.
• Reaction: Hydrolysis of the 1,4-beta-linkages of N-acetyl-D-glucosamine polymers of chitin.
• Enzyme class 2: E.C.3.2.1.17 - Lysozyme.
• Reaction: Hydrolysis of the 1,4-beta-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in peptidoglycan heteropolymers of the prokaryotes cell walls.

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

 Gene Ontology (GO) functional annotation 

• Cellular component: vacuole (1 term)
• Biological process: metabolic process (4 terms)
• Biochemical function: catalytic activity (7 terms)

DOI no: 10.1046/j.0014-2956.2001.02721.x

Eur J Biochem 269:893-901 (2002)

PubMed id: 11846790

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis.

E.Bokma, H.J.Rozeboom, M.Sibbald, B.W.Dijkstra, J.J.Beintema.

ABSTRACT

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower k(cat) and a slightly higher K(m) than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with approximately 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower k(cat) and an approximately twofold higher K(m) than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N-acetyl group of the -1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125 and Tyr183 contribute to catalysis by positioning the carbonyl oxygen of the N-acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis.

Selected figure(s)

Figure 3.
Fig. 3. Stereo representation of (A ) wild-type hevamine complexed with the degradation product chitotetraose in the active site [ 14 ], compared with (B ) the Asp125Ala/Glu127Ala and (C) the Asp125Ala/Tyr183Phe double mutants with bound chitotetraose. Only the carbohydrate residue bound at subsite -1 is shown. Hydrogen bonds are indicated with dashed lines. In wild-type hevamine, the oxygen atom of the N- acetyl group of the -1 sugar is positioned close to the C1 atom of the -1 sugar, and is hydrogen bonded to Tyr183. Asp125 makes a hydrogen bond to the nitrogen atom of the N -acetyl group. In the double mutants, the N -acetyl group points away from the C1 atom, and its hydrogen bonding interactions are lost. In addition, in the Asp125Ala/Tyr183Phe mutant, the Glu127 side chain has rotated away from the scissile bond glycosidic oxygen and is therefore in a less favourable position for its function as catalytic acid. HOH in Fig. 3B Go-is a well-defined water molecule. This figure was made with the program molscript[32].

Figure 4.
Fig. 4. Stabilization of the putative oxazolinium ion reaction intermediate. Hydrogen bonding interactions with Asp125 and Tyr183 are indicated.

The above figures are reprinted by permission from the Federation of European Biochemical Societies: Eur J Biochem (2002, 269, 893-901) copyright 2002.

Figures were selected by an automated process.