PDBsum entry: 1e6r

Hydrolase

PDB-id: 1e6r

Biological unit* = asymmetric unit, as shown
(*as deduced by PQS)

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Description

Header details

Header records

References

PROCHECK

Protein chains

496 a.a. *

Ligands

NAA-NAA-AMI ×2

SO4 ×4

Waters ×350

* Residue conservation analysis

Tools

Clefts Calculation

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PDB id:

1e6r

Name:

Hydrolase

Title:

Chitinase b from serratia marcescens wildtype in complex with inhibitor allosamidin

Structure:

Chitinase b. Chain: a, b. Engineered: yes

Source:

Serratia marcescens. Organism_taxid: 615. Expressed in: escherichia coli. Expression_system_taxid: 562

Biological unit:

Dimer (from PQS)

UniProt:

Chains A, B: Q54276 (Q54276_SERMA)

Seq:

Struc:

Seq:

499 a.a.

Struc:

496 a.a.

Key:	PfamA domain
Secondary structure	CATH domain

Resolution:

2.50Å

R-factor:

0.198

R-free:

0.255

Authors:

D.Komander,B.Synstad,V.G.H.Eijsink,D.M.F.Van Aalten

Key ref:

D.M.van Aalten et al. (2001). Structural insights into the catalytic mechanism of a family 18 exo-chitinase.. Proc Natl Acad Sci U S A, 98, 8979-8984. [PubMed id: 11481469] [DOI: 10.1073/pnas.151103798]

Date:

22-Aug-00

Release date:

22-Jun-01

Related entries:

1e15 chitinase b from serratia marcescens
1e6n chitinase b from serratia marcescens inactive mutant e144q in complex with n-acetylglucosamine-pentamer
1e6p chitinase b from serratia marcescens inactive mutant e144q

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Clefts

Surface

Key reference

DOI no: 10.1073/pnas.151103798

Proc Natl Acad Sci U S A 98:8979-8984 (2001)

PubMed id: 11481469

Structural insights into the catalytic mechanism of a family 18 exo-chitinase.

D.M.van Aalten, D.Komander, B.Synstad, S.Gåseidnes, M.G.Peter, V.G.Eijsink.

ABSTRACT

Chitinase B (ChiB) from Serratia marcescens is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites -2 to +3) shows closure of the "roof" of the active site tunnel. It also shows that the sugar in the -1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.

Selected figure(s)

Figure 1.
Fig. 1. Proposed catalytic mechanism. Asp-140, Asp-142, and Glu-144, conserved in most family 18 chitinases, are shown during separate stages of catalysis. The mechanism is based on proposals by Tews et al. (9) and Brameld and Goddard (15) and refined/expanded on the basis of the results presented in this paper. A three-dimensional view of the changing interactions in the crystal structures described here is shown in Fig. 2. (A) Resting enzyme. Asp-142 is too far away to interact with Glu-144. (B) Binding of substrate (only 1 binding NAG residue is shown) causes distortion of the pyranose ring to a boat or skewed boat conformation (see also Fig. 2) and rotation of Asp-142 toward Glu-144, enabling hydrogen bond interactions between the hydrogen of the acetamido group, Asp-142, and Glu-144. (C) Hydrolysis of the oxazolinium ion intermediate leads to protonation of Glu-144 and rotation of Asp-142 to its original position where it shares a proton with Asp-140.

Figure 2.
Fig. 2. Structures of the ChiB complexes. The EQ, EQ_NAG5, WT_ALLO, and WT_RX structures are shown as stereo images in the sequence as they would occur along the reaction coordinate (see also Fig. 1). In the stereo images, side chains (carbons in black) interacting with the sugars are shown as sticks, together with relevant stretches of backbone (gray). The sugars are drawn in a stick model with green carbons. Water molecules discussed in the text are shown as blue spheres. Unbiased F[o] F[c] maps (i.e., before inclusion of any ligand atom) are contoured at 2.25 [magenta, except for the uninterpreted density at 1 in WT_RX (yellow)]. Hydrogen bonds discussed in the text are drawn as dotted lines. Labels identify amino acid side chains in EQ, and the sugars bound to subsites 2 to +3 in EQ_NAG5.

Figures were selected by an automated process.