PDBsum entry 2anh

Alkaline phosphatase

PDB id: 2anh

Contents

Protein chains

446 a.a. *

Ligands

PO4 ×3

Metals

_ZN ×6

Waters ×185

* Residue conservation analysis

PDB id:

2anh

Name:

Alkaline phosphatase

Title:

Alkaline phosphatase (d153h)

Structure:

Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes. Other_details: 65% saturating (nh4)2so4, 100 mm tris, 10 mm mgcl2, 10 mm zncl2, 2 mm nah2po4 at ph 7.5

Source:

Escherichia coli. Organism_taxid: 562. Strain: ek1457. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.40Å R-factor: 0.185 R-free: 0.239

Authors:

J.E.Murphy,T.T.Tibbitts,E.R.Kantrowitz

Key ref:

J.E.Murphy et al. (1995). Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases. J Mol Biol, 253, 604-617. PubMed id: 7473737 DOI: 10.1006/jmbi.1995.0576

Date:

06-Sep-95 Release date: 29-Jan-96

Supersedes:

1anh

Protein chains

P00634  (PPB_ECOLI) -  Alkaline phosphatase from Escherichia coli (strain K12)

Seq: 471 a.a.

Struc: 446 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.1.3.1 - alkaline phosphatase.
• Reaction: a phosphate monoester + H2O = an alcohol + phosphate

phosphate monoester + H2O = alcohol + phosphate (Bound ligand (Het Group name = PO4) corresponds exactly)

• Cofactor: Mg(2+); Zn(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1006/jmbi.1995.0576

J Mol Biol 253:604-617 (1995)

PubMed id: 7473737

Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.

J.E.Murphy, T.T.Tibbitts, E.R.Kantrowitz.

ABSTRACT

In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E. coli alkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast and E. coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155. The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes. The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328. In the E. coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations. The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures. In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site. The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes.

Selected figure(s)

Figure 2.
Figure 2. The active site region of E. coli alkaline phosphatase including the phosphate group, magnesium ion and two zinc-binding sites. Not all the ligands are shown. Water molecules are indicated by the letter w. Hydrogen bonds are shown as broken lines (Kim & Wyckoff, 1991).

Figure 9.
Figure 9. Stereoview comparing the wild-type (thinnest lines), the K328H (middle lines) and the DM (thickest lines) structures. This view focuses on the new anion binding site (PO4-B) around Tyr169. This site exists in the D153H and DM structures, but is not seen in the K328H or the wild-type structure. The Figure shows the loop region formed by the disulfide bridge between Cys168 and Cys178. The conformation of this loop region appears to be conserved in all three structures.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1995, 253, 604-617) copyright 1995.

Figures were selected by an automated process.