PDBsum entry 1ux1

Hydrolase

PDB id: 1ux1

Contents

Protein chains

131 a.a. *

Ligands

THU ×4

TRS

Metals

_ZN ×4

Waters ×98

* Residue conservation analysis

PDB id:

1ux1

Name:

Hydrolase

Title:

Bacillus subtilis cytidine deaminase with a cys53his and an arg56gln substitution

Structure:

Cytidine deaminase. Chain: a, b, c, d. Synonym: cytidine aminohydrolase, cda. Engineered: yes. Mutation: yes

Source:

Bacillus subtilis. Organism_taxid: 1423. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.36Å R-factor: 0.213 R-free: 0.245

Authors:

E.Johansson,J.Neuhard,M.Willemoes,S.Larsen

Key ref:

E.Johansson et al. (2004). Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase. Biochemistry, 43, 6020-6029. PubMed id: 15147186 DOI: 10.1021/bi035893x

Date:

18-Feb-04 Release date: 20-May-04

Protein chains

P19079  (CDD_BACSU) -  Cytidine deaminase from Bacillus subtilis (strain 168)

Seq: 136 a.a.

Struc: 131 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.5.4.5 - cytidine deaminase.
• Reaction:
  1. cytidine + H2O + H⁺ = uridine + NH4⁺
  2. 2'-deoxycytidine + H2O + H⁺ = 2'-deoxyuridine + NH4⁺

cytidine + H2O + H(+) = uridine + NH4(+) (Bound ligand (Het Group name = THU) matches with 94.12% similarity)

2'-deoxycytidine + H2O + H(+) = 2'-deoxyuridine (Bound ligand (Het Group name = THU) corresponds exactly) + NH4(+)

• Cofactor: Zn(2+)

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

reference

DOI no: 10.1021/bi035893x

Biochemistry 43:6020-6029 (2004)

PubMed id: 15147186

Structural, kinetic, and mutational studies of the zinc ion environment in tetrameric cytidine deaminase.

E.Johansson, J.Neuhard, M.Willemoës, S.Larsen.

ABSTRACT

The zinc-containing cytidine deaminase (CDA, EC 3.5.4.5) is a pyrimidine salvage enzyme catalyzing the hydrolytic deamination of cytidine and 2'-deoxycytidine forming uridine and 2'-deoxyuridine, respectively. Homodimeric CDA (D-CDA) and homotetrameric CDA (T-CDA) both contain one zinc ion per subunit coordinated to the catalytic water molecule. The zinc ligands in D-CDA are one histidine and two cysteine residues, whereas in T-CDA zinc is coordinated to three cysteines. Two of the zinc coordinating cysteines in T-CDA form hydrogen bonds to the conserved residue Arg56, and this residue together with the dipole moments from two alpha-helices partially neutralizes the additional negative charge in the active site, leading to a catalytic activity similar to D-CDA. Arg56 has been substituted by a glutamine (R56Q), the corresponding residue in D-CDA, an alanine (R56A), and an aspartate (R56D). Moreover, one of the zinc-liganding cysteines has been substituted by histidine to mimic D-CDA, alone (C53H) and in combination with R56Q (C53H/R56Q). R56A, R56Q, and C53H/R56Q contain the same amount of zinc as the wild-type enzyme. The zinc-binding capacity of R56D is reduced. Only R56A, R56Q, and C53H/R56Q yielded measurable CDA activity, R56A and R56Q with similar K(m) but decreased V(max) values compared to wild-type enzyme. Because of dissociation into its inactive subunits, it was impossible to determine the kinetic parameters for C53H/R56Q. R56A and C53H/R56Q display increased apparent pK(a) values compared to the wild-type enzyme and R56Q. On the basis of the structures of R56A, R56Q, and C53H/R56Q an explanation is provided of kinetic results and the apparent instability of C53H/R56Q.