PDBsum entry: 2him

Hydrolase

PDB-id: 2him

Contents

Description

Header details

Header records

References

PROCHECK

Protein chains

324 a.a. *

Ligands

ASP ×4

ASN ×8

EDO ×8

Waters ×515

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

2him

Name:

Hydrolase

Title:

Crystal structure and allosteric regulation of the cytoplasmic escherichia coli l-asparaginase i

Structure:

L-asparaginase 1. Chain: a, b, c, d. Synonym: l-asparaginase i, l-asparagine amidohydrolase i, l-asnase i. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: ansa. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

UniProt:

Chains A, B, C, D: P0A962 (ASPG1_ECOLI)

Seq:

Struc:

Seq:

338 a.a.

Struc:

324 a.a.*

Key:

PfamA domain

Secondary structure

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme class:

E.C.3.5.1.1   [IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Reaction:

L-asparagine + H₂O = L-aspartate + NH₃ (see diagram below)

Resolution:

1.82Å

R-factor:

0.209

R-free:

0.226

Authors:

M.K.Yun,A.Nourse,S.W.White,C.O.Rock,R.J.Heath

Key ref:

M.K.Yun et al. (2007). Crystal structure and allosteric regulation of the cytoplasmic Escherichia colil-asparaginase I.. J Mol Biol, 369, 794-811. [PubMed id: 17451745] [DOI: 10.1016/j.jmb.2007.03.061]

Date:

29-Jun-06

Release date:

15-May-07

Related entries:

2p2d
2p2n

Quick_links

Procheck

Surface

Key reference

DOI no: 10.1016/j.jmb.2007.03.061

J Mol Biol 369:794-811 (2007)

PubMed id: 17451745

Crystal structure and allosteric regulation of the cytoplasmic Escherichia colil-asparaginase I.

M.K.Yun, A.Nourse, S.W.White, C.O.Rock, R.J.Heath.

ABSTRACT

AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is positively cooperative, displaying a sigmoidal substrate dependence curve with an [S](0.5) of 1 mM L-asparagine and a Hill coefficient (n(H)) of 2.6. Binding of L-asparagine to an allosteric site was observed in the crystal structure concomitant with a reorganization of the quarternary structure, relative to the apo enzyme. The carboxyl group of the bound asparagine makes salt bridges and hydrogen bonds to Arg240, while the N(delta2) nitrogen interacts with Thr162. Mutation of Arg240 to Ala increases the [S](0.5) value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity. Mutation of Thr162 to Ala results in an active enzyme with no cooperativity. Transmission of the signal from the allosteric site to the active site appears to involve subtle interactions at the dimer-dimer interface and relocation of Gln118 into the vicinity of the active site to position the probable catalytic water molecule. These data define the structural basis for the cooperative regulation of the intracellular asparaginase that is required for proper functioning within the cell.

Selected figure(s)

Figure 4.
Figure 4. The AnsA allosteric asparagine binding pocket. (a) The pocket is at the tight dimer interface (between monomers A and C in this view) at the N terminus of helix α8 and spanning dyad-related arginine residues 240 and 240′. Details of the interaction are provided in the text. Elements of monomer A and C are shown in yellow and cyan, respectively. Note that two dyad-related pockets are visible in this view, as well as the active site aspartate in monomer A adjacent to Thr91. Note also that Thr162 and Lys163 connect the allosteric and active sites within one monomer. (b) Diagrammatic representation of the hydrogen bonding interactions of asparagine (brown bonds) in the allosteric site (yellow bonds). The Figure was generated with LIGPLOT.^58

Figure 6.
Figure 6. Stereo views of the AnsA active site. (a) The apo enzyme (APOM). (b) The AnsA–asparagine complex showing the covalently attached product aspartate (green) at the active site. Water molecule W2 hydrogen bonded to Thr91 and Gln118 is ideally positioned to act as the nucleophile that will release the product (green). (c) The AnsA–asparagine complex showing asparagine (green) bound in the non-productive alternate conformation. In each panel, monomer A is in yellow, B is in green and C is in cyan. Details of these interactions are provided in the text, but the key active site residues are Thr14, Thr91, Lys163 and Asp92. Note how Gln118 changes conformation as the tetramer is compacted in (b) versus (a). Note also the pseudo-mirror symmetry in the active site that accommodates the two alternate binding modes in (b) and (c). Large purple dots indicate a salt bridge, while small, light gray dots indicate missing structural elements. (d) Electron density observed in the active site that was interpreted as overlapped aspartate and asparagine in the non-productive alternate conformation. The F[o]–F[c] simulated annealing omit map is displayed at a contour level of 4σ (blue).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 369, 794-811) copyright 2007.

Figures were selected by the author.