Human cytidine deaminase: A three-dimensional homology model of a tetrameric metallo-enzyme inferred from the crystal structure of a distantly related dimeric homologue

Costanzi, Stefano; Vincenzetti, Silvia; Cristalli, Gloria; Vita, Alberto

doi:10.1016/j.jmgm.2005.10.008

Cited by 17 publications

(28 citation statements)

References 17 publications

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“…Successively the crystal structure of the tetrameric B. subtilis CDA evidenced that a complicate set of intersubunit interactions contributes to the building of the active site in each monomer [4]. This structure, as well as earlier molecular models of the human CDA [2] based on the E. coli enzyme [3], indicated that an essential zinc ion is tetrahedrally coordinated by the uridine O-4 and the thiolate side chains of Cys65, Cys99, and Cys102. Another important residue involved in the catalytic mechanism is Glu 67 that would be located in proximity of the zinc ion and the positions 4 and 3 of the nucleoside pyrimidine ring.…”

Section: 1 Introductionmentioning

confidence: 92%

“…The four active sites, which lie at the subunit interface, operate independently and each subunit is equipped with a zinc atom that is involved in the covalent addition of water to cytidine, followed by elimination of ammonia [1, 2]. The structure of the CDA active site was first elucidated by Betts et al [3] crystallizing the dimeric E. coli enzyme.…”

Section: 1 Introductionmentioning

confidence: 99%

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Role of tyrosine 33 residue for the stabilization of the tetrameric structure of human cytidine deaminase

Micozzi¹,

Pucciarelli²,

Carpi³

et al. 2010

International Journal of Biological Macromolecules

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In the present work the effect of a mutation on tyrosine 33 residue (Y33G) of human cytidine deaminase (CDA) was investigated with regard to protein solubility and specific activity. Osmolytes and CDA ligands were used to increase the yield and the specific activity of the protein. The mutant enzyme was purified and subjected to a kinetic characterization and to stability studies. These investigations reinforced the hypothesis that in human CDA the side chain of Y33 is involved in intersubunit interactions with four glutamate residues (E108) forming a double latch that connects each of the two pairs of monomers of the tetrameric CDA.Keywords cytidine deaminase; site-directed mutagenesis; molecular modeling; circular dichroism IntroductionCytidine deaminase (CDA, cytidine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5), plays an important role in the pyrimidine salvage pathways since it is involved in the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. This Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptInt J Biol Macromol. Author manuscript; available in PMC 2011 November 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript enzyme also catalyzes the deamination of cytosine nucleoside analogues used in chemotherapy leading to the loss of their pharmacological activity.Human cytidine deaminase consists of four identical subunits (4 × 14,900 Da) with one active site per subunit. The four active sites, which lie at the subunit interface, operate independently and each subunit is equipped with a zinc atom that is involved in the covalent addition of water to cytidine, followed by elimination of ammonia [1,2]. The structure of the CDA active site was first elucidated by Betts et al. [3] crystallizing the dimeric E. coli enzyme. In this protein the active site of the monomer is formed with the contribution of residues coming from the so called "broken active site". Successively the crystal structure of the tetrameric B. subtilis CDA evidenced that a complicate set of intersubunit interactions contributes to the building of the active site in each monomer [4]. This structure, as well as earlier molecular models of the human CDA [2] based on the E. coli enzyme [3], indicated that an essential zinc ion is tetrahedrally coordinated by the uridine O-4 and the thiolate side chains of Cys65, Cys99, and Cys102. Another important residue involved in the catalytic mechanism is Glu 67 that would be located in proximity of the zinc ion and the positions 4 and 3 of the nucleoside...

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Section: 1 Introductionmentioning

confidence: 92%

Section: 1 Introductionmentioning

confidence: 99%

Role of tyrosine 33 residue for the stabilization of the tetrameric structure of human cytidine deaminase

Micozzi¹,

Pucciarelli²,

Carpi³

et al. 2010

International Journal of Biological Macromolecules

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“…The enzyme is active only as an intact tetramer, since three different monomers concur to the formation of each of the four active sites. [1–5] For example, the active site enclosed within subunit A1 is also lined by residues of subunits B1 and B2 (Figure 1). …”

Section: Introductionmentioning

confidence: 99%

“…In particular, our docking experiments targeted the crystal structure of the murine enzyme crystallized in complex with cytidine (PDB ID: 2FR6), [8] which, within the active site, shares 95% of sequence identity with its human homologue (Figure 1) – overall, human and murine CDA share a sequence identity of 81.5% and have a distance of 21.5 PAM (percentage of accepted mutations). [1] The only non conserved residue within a radius of 5 Å from the bound nucleosides is residue 64, which is an isoleucine in the human and a valine in the mouse enzyme (Figure 1). However, the sidechain of Ile/Val64 points away from the active site and is not involved in interactions with the ligands.…”

Section: Introductionmentioning

confidence: 99%

Delineation of the Molecular Mechanisms of Nucleoside Recognition by Cytidine Deaminase through Virtual Screening

Costanzi

Vilar

Micozzi³

et al. 2011

ChemMedChem

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Cytidine deaminase (3.5.4.5, CDA), an enzyme of the pyrimidine salvage pathways, is responsible for the degradation and inactivation of several cytidine-based antitumoral drugs, such as cytarabine, gemcitabine, decitabine, and azacytidine. Thus, CDA inhibitors are highly sought after as compounds to be co-administered with said drugs in order to improve their effectiveness. Alternatively, the design of antitumoral drugs not susceptible to the action of CDA is also regarded as an attractive solution. Here, we describe a virtual screening for CDA ligands based on chemical similarity and molecular docking. The campaign led to identification of three novel inhibitors and one novel substrate, with a 19% hit rate, and allowed a significant extension of the structure-activity relationships, also in light of the compounds that resulted inactive. The most active compound identified through the screening was the inhibitor pseudoisocytidine, which has the potential to serve as a lead for highly stable compounds. The study also delineated the detrimental effect of 5-aza and 6-aza substitutions, the incompatibility of the presence of an amino group at the 3′-position, as well as the presence of very strict steric requirements around the 2′-arabino position and, even more, the N4-position. Importantly, these features can be exploited for the design of novel antineoplastic agents resistant to the action of CDA.

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“…According to the high similarity of CDA amino acid sequences and structure between the bighead carp and Bacillus subtilis, the function of individual amino acid residues of bighead carp CDA could be easily deduced (Costanzi et al, 2006). Residues Cys71, Cys105 and Cys108 served as ligands for the zinc ion, and Glu73 functioned as proton donor and acceptor in catalysis.…”

Section: Cdna Sequence and Protein Analysis Of Bighead Carp Cytidine mentioning

confidence: 99%

Identification of cda gene in bighead carp and its expression in response to microcystin-LR

Cai

Liang

et al. 2012

Ecotoxicology and Environmental Safety

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Human cytidine deaminase: A three-dimensional homology model of a tetrameric metallo-enzyme inferred from the crystal structure of a distantly related dimeric homologue

Cited by 17 publications

References 17 publications

Role of tyrosine 33 residue for the stabilization of the tetrameric structure of human cytidine deaminase

Role of tyrosine 33 residue for the stabilization of the tetrameric structure of human cytidine deaminase

Delineation of the Molecular Mechanisms of Nucleoside Recognition by Cytidine Deaminase through Virtual Screening

Identification of cda gene in bighead carp and its expression in response to microcystin-LR

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