PDBsum entry: 1ll4

Hydrolase

PDB id: 1ll4

Contents

Protein chains

392 a.a. *

Ligands

NAA-NAA-AMI ×4

Waters ×100

* Residue conservation analysis

PDB id:

1ll4

Name:

Hydrolase

Title:

Structure of c. Immitis chitinase 1 complexed with allosamidin

Structure:

Chitinase 1. Chain: a, b, c, d. Fragment: residues 36-427. Engineered: yes

Source:

Coccidioides immitis. Organism_taxid: 5501. Gene: cts1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.80Å R-factor: 0.197 R-free: 0.258

Authors:

K.Bortone,A.F.Monzingo,S.Ernst,J.D.Robertus

Key ref:

K.Bortone et al. (2002). The structure of an allosamidin complex with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-assisted mechanism. J Mol Biol, 320, 293-302. PubMed id: 12079386 DOI: 10.1016/S0022-2836(02)00444-8

Date:

26-Apr-02 Release date: 25-Sep-02

Protein chains

P0CB51  (CHI1_COCPO) -  Endochitinase 1

Seq: 427 a.a.

Struc: 392 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.14 - Chitinase.
• Reaction: Hydrolysis of the 1,4-beta-linkages of N-acetyl-D-glucosamine polymers of chitin.

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (4 terms)
• Biochemical function: catalytic activity (7 terms)

DOI no: 10.1016/S0022-2836(02)00444-8

J Mol Biol 320:293-302 (2002)

PubMed id: 12079386

The structure of an allosamidin complex with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-assisted mechanism.

K.Bortone, A.F.Monzingo, S.Ernst, J.D.Robertus.

ABSTRACT

Allosamidin is a known inhibitor of class 18 chitinases. We show that allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from Coccidioides immitis, with a K(i) of 60 nM. We report the X-ray structure of the complex and show that upon inhibitor binding the side-chain of Asp169 rotates to form an ion pair with the oxazolinium cation. The mechanism of action is thought to involve protonation of the leaving group by Glu171 and substrate assistance by the sugar acetamido moiety to form an oxazoline-like intermediate. We converted both amino acid residues to the corresponding amide and found that each mutation effectively abolishes enzyme activity. X-ray structures show the mutant enzymes retain the basic wild-type structure and that the loss of mutant activity is due to their altered chemical properties. The high affinity of allosamidin, and its similarity to the putative reaction intermediate, suggests it is a transition state analog. This helps validate our contention that the role of Asp169 is to electrostatically stabilize the reaction transition state.

Selected figure(s)

Figure 6.
Figure 6. The active site of wild-type and mutant CiX1. The conformation of key active-site residues of the wild-type enzyme are shown as thin solid bonds. For reference, the position of the allosamizoline ring of allosamidin is shown in dashed lines. The conformation of the C^a trace for residues 168-172, and the side-chains of 169 and 171 for the E171Q mutant is shown as thick black bonds. The conformation of corresponding atoms of the D169N mutant is shown as thick gray bonds.

Figure 7.
Figure 7. Proposed mechanism of catalysis. The first panel, upper left, shows the arrangement of key active-site residues and a conventional b-linked saccharide. As the substrate is bound, in the upper right panel, the polysaccharide chain is rotated and bent between sugars at sites -1 and +1, facilitating a distortion of the sugar at site -1 towards a high energy boat conformation.13 Two hydrogen bonds are formed with the site -1 sugar: one between a water bound to Tyr239 and the acetoamido nitrogen atom of the sugar, and the other between the sugar O6 atom and the side-chain of Asp240. A hydrogen bond is also formed between the protonated E171 side-chain and the oxygen atom of the scissile glycolytic bond. As protonation of the leaving group (+1 site sugar) proceeds and the glycosidic bond between the sugars at -1 and +1 sites breaks, the sugar at -1 develops cationic character. This cationic character is stabilized by interaction with the -1 acetamido group, and a positively charged oxazolinium intermediate is formed. The side-chain of Asp169 rotates to form a stabilizing ion pair interaction with the cationic intermediate. A water, which may be the one displaced by the oxazolinium intermediate, then attacks the C1 atom of the intermediate resulting in the formation of the b-anomer.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 320, 293-302) copyright 2002.

Figures were selected by an automated process.