Re‐evaluation of the colorimetric assay for cytidine deaminase activity

Okamura, Takashi; Kigasawa, K.

doi:10.1002/pd.1970140313

Cited by 6 publications

(4 citation statements)

References 15 publications

(11 reference statements)

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“…Product identities were further confirmed by UV spectroscopy of the eluting materials. Ammonia concentrations were determined using the Berthelot method as previously described (22). Standard melamine was obtained from Sigma-Aldrich (St. Louis, MO), and cyanuric acid was from Fluka (Buchs, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

Bacterial Ammeline Metabolism via Guanine Deaminase

et al. 2010

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Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.

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Section: Methodsmentioning

confidence: 99%

Bacterial Ammeline Metabolism via Guanine Deaminase

et al. 2010

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“…CDA activity was measured spectrophotometrically as previously described 25,35,36 using a simple and rapid high-performance liquid chromatography-based method derived from Okamura et al After overnight incubation of serum samples, CDA activity was evaluated on the conversion of cytidine to uridine with ammonium release. CDA activity was finally expressed as units per milligram proteins, with 1 U = 10 -4 mmol ammonium released/min.…”

Section: Cytidine Deaminase Phenotypic Assaymentioning

confidence: 99%

High-Resolution Melting Analysis of Sequence Variations in the Cytidine Deaminase Gene (CDA) in Patients With Cancer Treated With Gemcitabine

Raynal¹,

Ciccolini²,

Mercier³

et al. 2010

Therapeutic Drug Monitoring

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Gemcitabine (2',2'-difluorodeoxycytidine) is a major antimetabolite cytotoxic drug with a wide spectrum of activity against solid tumors. Hepatic elimination of gemcitabine depends on a catabolic pathway through a deamination step driven by the enzyme cytidine deaminase (CDA). Severe hematologic toxicity to gemcitabine was reported in patients harboring genetic polymorphisms in CDA gene. High-resolution melting (HRM) analysis of polymerase chain reaction amplicon emerges today as a powerful technique for both genotyping and gene scanning strategies. In this study, 46 DNA samples from gemcitabine-treated patients were subjected to HRM analysis on a LightCycler 480 platform. Residual serum CDA activity was assayed as a surrogate marker for the overall functionality of this enzyme. Genotyping of three well-described single nucleotide polymorphisms in coding region (c.79A>C, c.208G>A and c.435C>T) was successfully achieved by HRM analysis of small polymerase chain reaction fragments, whereas unknown single nucleotide polymorphisms were searched by a gene scanning strategy with longer amplicons (up to 622 bp). The gene scanning strategy allowed us to find a new intronic mutation c.246+37G>A in a female patient displaying marked CDA deficiency and who had an extreme toxic reaction with a fatal outcome to gemcitabine treatment. Our work demonstrates that HRM-based methods, owing to their simplicity, reliability, and speed, are useful tools for diagnosis of CDA deficiency and could be of interest for personalized medicine.

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“…Catalysis was stopped by boiling reaction tubes for 10 min, tubes were cooled to room temperature, and 3 g of purified biuret hydrolase (12) was added. After 1 h, ammonia concentrations were determined by using the Berthelot reaction (36). Products produced by enzymatic reactions were monitored by nuclear magnetic resonance (NMR) spectroscopy using universally labeled […”

Section: Methodsmentioning

confidence: 99%

Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

Dodge

Preiner

Wackett

2013

Journal of Bacteriology

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The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13 C nuclear magnetic resonance ( 13 C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria.

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Re‐evaluation of the colorimetric assay for cytidine deaminase activity

Cited by 6 publications

References 15 publications

Bacterial Ammeline Metabolism via Guanine Deaminase

Bacterial Ammeline Metabolism via Guanine Deaminase

High-Resolution Melting Analysis of Sequence Variations in the Cytidine Deaminase Gene (CDA) in Patients With Cancer Treated With Gemcitabine

Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

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