PDBsum entry 1a5l

Hydrolase

PDB id: 1a5l

Contents

Protein chains

100 a.a. *

101 a.a. *

536 a.a. *

Waters ×183

* Residue conservation analysis

PDB id:

1a5l

Name:

Hydrolase

Title:

K217c variant of klebsiella aerogenes urease

Structure:

Urease (gamma subunit). Chain: a. Engineered: yes. Mutation: yes. Urease (beta subunit). Chain: b. Engineered: yes. Urease (alpha subunit). Chain: c.

Source:

Klebsiella aerogenes. Organism_taxid: 28451. Gene: urea, ureb, urec. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PDB file)

Resolution:

2.20Å R-factor: 0.186

Authors:

M.A.Pearson,R.A.Schaller,L.O.Michel,P.A.Karplus,R.P.Hausinger

Key ref:

M.A.Pearson et al. (1998). Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand. Biochemistry, 37, 6214-6220. PubMed id: 9558361 DOI: 10.1021/bi980021u

Date:

17-Feb-98 Release date: 27-May-98

Protein chain

P18316  (URE3_KLEAE) -  Urease subunit gamma from Klebsiella aerogenes

Seq: 100 a.a.

Struc: 100 a.a.

Protein chain

P18315  (URE2_KLEAE) -  Urease subunit beta from Klebsiella aerogenes

Seq: 106 a.a.

Struc: 101 a.a.

Protein chain

P18314  (URE1_KLEAE) -  Urease subunit alpha from Klebsiella aerogenes

Seq: 567 a.a.

Struc: 536 a.a.*

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

Enzyme reactions

• Enzyme class: Chains A, B, C: E.C.3.5.1.5 - urease.
• Reaction: urea + 2 H2O + H⁺ = hydrogencarbonate + 2 NH4⁺
• Cofactor: Ni(2+)

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

Added reference

DOI no: 10.1021/bi980021u

Biochemistry 37:6214-6220 (1998)

PubMed id: 9558361

Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand.

M.A.Pearson, R.A.Schaller, L.O.Michel, P.A.Karplus, R.P.Hausinger.

ABSTRACT

Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.