期刊
INTERNATIONAL JOURNAL OF PEPTIDE RESEARCH AND THERAPEUTICS
卷 21, 期 3, 页码 365-374出版社
SPRINGER
DOI: 10.1007/s10989-015-9465-9
关键词
Bioinformatics analysis; Escherichia coli; Cytosine deaminase; Enzyme/prodrug
资金
- Research Council of Shiraz University of Medical Sciences, Shiraz, Iran
Cytosine deaminase (CDase) catalyzes the deamination of cytosine and 5-fluorocytosine (5-FC) to uracil and 5-fluorouracil (5-FU), respectively. The enzyme is used for cancer therapy, as cancerous cells are not able to synthesize cytosine via a de-novo pathway. In order to characterize the Escherichia coli AGH09 CDase, a strong CDase producing strain, was isolated from the urinary infection patients. The DNA fragment encoding the CDase was cloned into pET22b vector and sequenced. The presented paper uses bioinformatics and experimental approaches to describe the physiochemical, functional and structural properties of CDase. The results revealed that the enzyme was active in pH ranging from 6.0 to 9.0 along with an optimal activity at 47 A degrees C. Moreover, in the presence of Cu++, Zn++ and Fe++ ions, the enzyme activity was increased; however, the Mn++ and Na+ ions had negatively influence on CDase activity. Bioinformatics analysis showed the enzyme had no signal sequences and disulfide bond along with no allergenicity. The aliphatic index (94.59) and grand average of hydropathicity index (-0.21) calculated by ProtParam server indicated that CDase may be stable for a wide range of temperature and has good solubility in water. Secondary structure analysis revealed that most of the recombinant CDase has predominant alpha-helical structures (37.24 %) and Random coils (39.58 %). The three dimensional structure of CDase is predicted by homology modeling using the known CDase crystal structures of E. coli (3O7U) as template using Geno3D2 Web Server. The model was evaluated with PROCHECK and WHAT IF programs. These findings suggested our enzyme might be a novel CDase for deployment in cancer treatment. Moreover, our data provides such beneficial information for a better drug formulation with proper storage shelf life storage.
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