Cytidine deaminase from Escherichia coli B. Purification and enzymic and molecular properties

Vita, Alberto; Amici, Adolfo; Cacciamani, Tiziana; Lanciotti, Marina; Magni, Giulio

doi:10.1021/bi00342a049

Cited by 20 publications

(11 citation statements)

References 21 publications

(13 reference statements)

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“…The enzyme kinetic parameters were then determined (Table 1) from double‐reciprocal plots of initial velocities vs substrate concentration (both 2′‐deoxycytidine and cytidine). The values obtained are in good agreement with values reported for the purified E. coli enzyme and for recombinant human and Brugia pahangi cytidine deaminases [18–21]. As the E. coli CDA, At‐CDA1 has a K m for cytidine about twofold higher than for 2′‐deoxycytidine (Table 1).…”

Section: Resultssupporting

confidence: 87%

A prokaryotic‐type cytidine deaminase from Arabidopsis thaliana

Faivre-Nitschke

Grienenberger

Gualberto

1999

European Journal of Biochemistry

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The gene and cDNA of an Arabidopsis thaliana cytidine deaminase (CDA) were cloned and sequenced. The gene, At-cda1, is located on chromosome 2 and is expressed in all plant tissues tested, although with quantitative differences. Expression analysis suggest that At-cda1 probably codes for the housekeeping cytidine deaminase of Arabidopsis. The gene was functionally expressed in Escherichia coli and the protein, At-CDA1, shows similar enzymatic and substrate specificities as conventional cytidine deaminases: it deaminates cytidine and deoxycytidine and is competitively inhibited by cytosine-containing compounds. Because the protein shows no affinity to RNA, it is not likely to be involved in RNA-editing by C-to-U deamination.When compared to cytidine deaminases from other organisms, it becomes clear that At-CDA1 is related, both in sequence and structure, to the CDA of E. coli and other gram-negative bacteria. The eubacterial nature of the Arabidopsis CDA suggests that it is an additional example of a plant gene of endosymbiotic origin.

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Section: Resultssupporting

confidence: 87%

A prokaryotic‐type cytidine deaminase from Arabidopsis thaliana

Faivre-Nitschke

Grienenberger

Gualberto

1999

European Journal of Biochemistry

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“…In 1994, Betts et al [7] published the first crystal structure of the E. coli D-CDA (PDB ID: 1CTU), which confirmed the homodimeric structure of the enzyme that we had previously hypothesized [4]. Each monomer, of a molecular weight of 31.5 kDa, was found to be composed of a small N-terminal alpha-helical domain with no evident function, a catalytic domain containing a zinc-binding pocket, and a C-terminal domain.…”

Section: Introductionmentioning

confidence: 58%

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

Vincenzetti

Cristalli

et al. 2006

Journal of Molecular Graphics and Modelling

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“…glycosidases and glycosyltransferases studies. It was consisIt should be noted that there is an essential SH-group in the tently shown that in these enzymes a carboxyl group mediates Upase molecule [2,11]. Despite the fact that in accordance with glycosyl transfer [20].…”

Section: ~ 310mentioning

confidence: 99%

“…alyzes the reversible phosphorolysis of uridine with the formation of riboso-l-phosphate and uracil [1,2]. The protein is involved in degradation of pyrimidine nucleosides and their util-2.…”

Section: Introductionmentioning

confidence: 99%

Atomic structure at 2.5 Å resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice

et al. 1995

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Received 18 april 1995 homology to the sequence of E. coli PNP -the sequence simiAbstract Uridine phosphorylase from E. coil (Upase) has been larity being 49% and the sequence identity 25% [9]. However crystallized using vapor diffusion technique in a new monoclinic the sequence homology between Upase and other phosphorycrystal form. The structure was determined by the molecular lases is very poor. The comparison of the three-dimensional replacement method at 2.5 ~. resolution. The coordinates of the trigonal crystal form were used as a starting model and the structures of Upase and other phosphorylases has shown that refinement by the program XPLOR led to the R-factor of 18.6%.the structure of Upase and human PNP are rather similar but The amino acid fold of the protein was found to be the same as there is no any similarity between Upase and thymidine that in the trigonal crystals. The positions of flexible regions were phosphorylase structures. The study of E. coli PNP is in prorefined. The conclusion about the involvement in the active site gress and obviously its structure must be similar to that of is in good agreement with the results of the biochemical experiUpase. The Upase three-dimensional structure has been solved ments, at 3.0 ~ resolution using the data from trigonal crystals described previously [9]. This structure contains some disordered Key words: Uridine phosphorylase; E. coli; X-ray structure regions and little is known about groups involved in catalysis and/or binding at the Upase active site [2,10 12]. IntroductionIn this paper we report results of an X-ray investigation of a monoclinic form at 2.5 ~ resolution and the interpretation of the data of the selective chemical modification of the essential Uridine phosphorylase (EC 2.4.2.3; Upase) from E. coli catamino acid residues. alyzes the reversible phosphorolysis of uridine with the formation of riboso-l-phosphate and uracil [1,2]. The protein is involved in degradation of pyrimidine nucleosides and their util-2. Materials and methods ization as carbon and energy sources in E. coli cells. Upase 2.1. Crystallization along with purine nucleoside phosphorylase (PNP) and thymid-The uridine phosphorylase was crystallized in different crystal forms ine phosphorylase, belongs to the class of nucleoside by the vapor diffusion technique at room temperature [13,14]. The phosphorylases. Uridine phosphorylase has been identified as monoclinic form was obtained in sitting drops of 0.05 M Tris-HC1 the enzyme which is responsible for the cleavage of some pyrimbuffer at pH 7.3 containing 10-12 mg/ml of the protein and 4-6% PEG (mol.wt. 4,000). The equilibrium solution consisted of 0.1 M Tris-mal/ idine nucleoside analogs possessing antitumor activity. ConverNaOH pH 5.91 5.96, 20-25% PEG (mol.wt. 4,000) and 0.04% sodium sion of 5-fluorouridine (FUR) and 5-fluorodeoxyuridine azide. The crystals reached the size of 0.7 × 0.3 x 0.5 mm after 4-6 (FUdR) to 5-fluorouracil (5FU) by Upase required the usage weeks. These crystals belong to monoclinic space group P2~...

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Cytidine deaminase from Escherichia coli B. Purification and enzymic and molecular properties

Cited by 20 publications

References 21 publications

A prokaryotic‐type cytidine deaminase from Arabidopsis thaliana

A prokaryotic‐type cytidine deaminase from Arabidopsis thaliana

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

Atomic structure at 2.5 Å resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice

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