PDBsum entry 1v51

Hydrolase

PDB id: 1v51

Contents

Protein chain

474 a.a. *

Ligands

ACT ×2

Metals

_ZN ×11

Waters ×255

* Residue conservation analysis

PDB id:

1v51

Name:

Hydrolase

Title:

The functional role of the binuclear metal center in d-aminoacylase. One-metal activation and second-metal attenuation

Structure:

D-aminoacylase. Chain: a. Engineered: yes

Source:

Alcaligenes faecalis. Organism_taxid: 511. Gene: da1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.60Å R-factor: 0.189 R-free: 0.208

Authors:

W.L.Lai,L.Y.Chou,C.Y.Ting,Y.C.Tsai,S.H.Liaw

Key ref:

W.L.Lai et al. (2004). The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation. J Biol Chem, 279, 13962-13967. PubMed id: 14736882 DOI: 10.1074/jbc.M308849200

Date:

20-Nov-03 Release date: 20-Apr-04

Protein chain

Q9AGH8  (Q9AGH8_ALCFA) -  D-aminoacylase from Alcaligenes faecalis

Seq: 484 a.a.

Struc: 474 a.a.

Enzyme reactions

• Enzyme class: E.C.3.5.1.81 - N-acyl-D-amino-acid deacylase.
• Reaction: an N-acyl-D-amino acid + H2O = a D-alpha-amino acid + a carboxylate

N-acyl-D-amino acid + H2O = D-alpha-amino acid (Bound ligand (Het Group name = ACT) matches with 60.00% similarity) + carboxylate

• Cofactor: Zn(2+)

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

Added reference

DOI no: 10.1074/jbc.M308849200

J Biol Chem 279:13962-13967 (2004)

PubMed id: 14736882

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation.

W.L.Lai, L.Y.Chou, C.Y.Ting, R.Kirby, Y.C.Tsai, A.H.Wang, S.H.Liaw.

ABSTRACT

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.

Selected figure(s)

Figure 3.
FIG. 3. The metal centers. A, the F[o] - F[c] electron density maps of the native enzyme in complex with 100 mM ZnCl[2] contoured at 15 level and shown in magenta, with 50 mM CdCl[2] contoured at 15 level and shown in cyan, and with 100 mM CuCl[2] contoured at 18 level and shown in green. The metal ligands are shown as a ball-and-stick representation, with the Zn2+ and Cu2+ ions as magenta and green spheres, respectively. Zn2+ and Cd^2+ bind at the subsite, where Cu2+ binds at the [2] subsite. B, the 2F[o] - F[c] electron density maps of the H220A mutant contoured at 2.5 level and shown in cyan, and the difference map for the zinc ion contoured at 15 level and shown in magenta. The endogenous zinc ion binds at the [3] subsite instead of the site in this mutant. C, the 2F[o] - F[c] electron density map of the D366A mutant contoured at 2.5 level and shown in cyan, and the difference map for the zinc ion in complex with 100 mM ZnCl[2] contoured at 15 level and shown in magenta. The additional zinc ion binds at the [4] subsite. D, superposition of the native enzyme with 100 mM ZnCl[2] in blue, the native enzyme with 100 CuCl[2] in green, the H220A mutant in yellow, and the D366A mutant with 100 mM ZnCl[2] in red. The different metal coordination is carried out by small shifts in the side chains of ligands and small movements of the metal ions.

Figure 4.
FIG. 4. The proposed mechanisms for catalysis (A) and metal attenuation (B). The numbers shown indicate the interatomic distances in angstroms. Asp366 maybe with assistance from His67 and His69, is responsible for the proton transfer from the water molecule to the amide nitrogen (3). The presence of the inhibitory metal ion at the [1] site might lower the pK[a] values of its ligand residues, His67, His69, and Asp366, and/or hold the active site water to perturb the proton shuttle and intermediate stabilization.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 13962-13967) copyright 2004.

Figures were selected by an automated process.