Guanosine Deaminase Research Articles

Binding asymmetry and conformational studies of the AtGSDA dimer

Guanosine deaminase (GSDA) is an important deaminase that converts guanosine to xanthosine, a key intermediate in nitrogen recycling in plants. We previously solved complex structures of Arabidopsis thaliana GSDA bound by various ligands and examined its catalytic mechanism. Here, we report cocrystal structures of AtGSDA bound by inactive guanosine derivatives, which bind relatively weakly to the enzyme and mostly have poor binding geometries. The two protomers display unequal binding performances, and molecular dynamics simulation identified diverse conformations during the enzyme-ligand interactions. Moreover, intersubunit, tripartite salt bridges show conformational differences between the two protomers, possibly acting as “gating” systems for substrate binding and product release. Our structural and biochemical studies provide a comprehensive understanding of the enzymatic behavior of this intriguing enzyme.

Substrate Specificity of GSDA Revealed by Cocrystal Structures and Binding Studies

In plants, guanosine deaminase (GSDA) catalyzes the deamination of guanosine for nitrogen recycling and re-utilization. We previously solved crystal structures of GSDA from Arabidopsis thaliana (AtGSDA) and identified several novel substrates for this enzyme, but the structural basis of the enzyme activation/inhibition is poorly understood. Here, we continued to solve 8 medium-to-high resolution (1.85-2.60 Å) cocrystal structures, which involved AtGSDA and its variants bound by a few ligands, and investigated their binding modes through structural studies and thermal shift analysis. Besides the lack of a 2-amino group of these guanosine derivatives, we discovered that AtGSDA's inactivity was due to the its inability to seclude its active site. Furthermore, the C-termini of the enzyme displayed conformational diversities under certain circumstances. The lack of functional amino groups or poor interactions/geometries of the ligands at the active sites to meet the precise binding and activation requirements for deamination both contributed to AtGSDA's inactivity toward the ligands. Altogether, our combined structural and biochemical studies provide insight into GSDA.

Enzymes and cellular interplay required for flux of fixed nitrogen to ureides in bean nodules

Tropical legumes transport fixed nitrogen in form of ureides (allantoin and allantoate) over long distances from the nodules to the shoot. Ureides are formed in nodules from purine mononucleotides by a partially unknown reaction network that involves bacteroid-infected and uninfected cells. Here, we demonstrate by metabolic analysis of CRISPR mutant nodules of Phaseolus vulgaris defective in either xanthosine monophosphate phosphatase (XMPP), guanosine deaminase (GSDA), the nucleoside hydrolases 1 and 2 (NSH1, NSH2) or xanthine dehydrogenase (XDH) that nodule ureide biosynthesis involves these enzymes and requires xanthosine and guanosine but not inosine monophosphate catabolism. Interestingly, promoter reporter analyses revealed that XMPP, GSDA and XDH are expressed in infected cells, whereas NSH1, NSH2 and the promoters of the downstream enzymes urate oxidase (UOX) and allantoinase (ALN) are active in uninfected cells. The data suggest a complex cellular organization of ureide biosynthesis with three transitions between infected and uninfected cells.

Initiation of cytosolic plant purine nucleotide catabolism involves a monospecific xanthosine monophosphate phosphatase

In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo.

The C-terminal loop of Arabidopsis thaliana guanosine deaminase is essential to catalysis.

Guanosine deaminase (GSDA) in plants specifically deaminates (de)guanosine to produce xanthosine with high specificity, which is further converted to xanthine, a key intermediate in purine metabolism and nitrogen recycling. We solved GSDA's structures from Arabidopsis thaliana in the free and ligand-bound forms at high resolutions. Unlike GDA, the enzyme employs a single-proton shuttle mechanism for catalysis and both the substrate and enzyme undergo structural rearrangements. The last fragment of the enzyme loops back and seals the active site, and the substrate rotates during the reaction, both essential to deamination. We further identified more substrates that could be employed by the enzyme and compare it with other deaminases to reveal the recognition differences of specific substrates. Our studies provide insight into this important enzyme involved in purine metabolism and will potentially aid in the development of deaminase-based gene-editing tools.

Активність ензимів катаболізму пуринових нуклеотидів у печінці щурів за умов аліментарного дисбалансу

The aim of the study was to investigate the activity of purine nucleotide catabolism enzymes, in particular, AMP-deaminase, 5'-nucleotidase, guanosine deaminase, and guanosine phosphorylase and xanthine oxidase in the cytosolic fraction of the liver of rats under conditions of different dietary supply of sucrose and dietary proteins. Enzyme activity was determined by photo colorimetric method: AMP-deaminase activity by the amount of ammonia formed by deamination of AMP, which has a maximum absorption at λ-540 nm and 5'-nucleotidase activity by the amount of Pn formed by hydrolysis of AMP at λ-8. The activity of guanosine phosphorylase, guanosine deaminase and xanthine oxidase was determined by spectrophotometric method. The results of studies have shown that due to consuming a high-sucrose diet in on the background of protein deficiency, the activation of purine nucleotide catabolism is observed and it can lead to disruption of the regulation of energy-dependent processes in liver cells. A critical factor influencing on the state of the purine nucleotide system and the activity of enzymes of their catabolism is alimentary protein deficiency.

Enhanced nucleotide analysis enables the quantification of deoxynucleotides in plants and algae revealing connections between nucleoside and deoxynucleoside metabolism.

Detecting and quantifying low-abundance (deoxy)ribonucleotides and (deoxy)ribonucleosides in plants remains difficult; this is a major roadblock for the investigation of plant nucleotide (NT) metabolism. Here, we present a method that overcomes this limitation, allowing the detection of all deoxy- and ribonucleotides as well as the corresponding nucleosides from the same plant sample. The method is characterized by high sensitivity and robustness enabling the reproducible detection and absolute quantification of these metabolites even if they are of low abundance. Employing the new method, we analyzed Arabidopsis thaliana null mutants of CYTIDINE DEAMINASE, GUANOSINE DEAMINASE, and NUCLEOSIDE HYDROLASE 1, demonstrating that the deoxyribonucleotide (dNT) metabolism is intricately interwoven with the catabolism of ribonucleosides (rNs). In addition, we discovered a function of rN catabolic enzymes in the degradation of deoxyribonucleosides in vivo. We also determined the concentrations of dNTs in several mono- and dicotyledonous plants, a bryophyte, and three algae, revealing a correlation of GC to AT dNT ratios with genomic GC contents. This suggests a link between the genome and the metabolome previously discussed but not experimentally addressed. Together, these findings demonstrate the potential of this new method to provide insight into plant NT metabolism.

AMP and GMP Catabolism in Arabidopsis Converge on Xanthosine, Which Is Degraded by a Nucleoside Hydrolase Heterocomplex.

Plants can fully catabolize purine nucleotides. A firmly established central intermediate is the purine base xanthine. In the current widely accepted model of plant purine nucleotide catabolism, xanthine can be generated in various ways involving either inosine and hypoxanthine or guanosine and xanthosine as intermediates. In a comprehensive mutant analysis involving single and multiple mutants of urate oxidase, xanthine dehydrogenase, nucleoside hydrolases, guanosine deaminase, and hypoxanthine guanine phosphoribosyltransferase, we demonstrate that purine nucleotide catabolism in Arabidopsis (Arabidopsis thaliana) mainly generates xanthosine, but not inosine and hypoxanthine, and that xanthosine is derived from guanosine deamination and a second source, likely xanthosine monophosphate dephosphorylation. Nucleoside hydrolase 1 (NSH1) is known to be essential for xanthosine hydrolysis, but the in vivo function of a second cytosolic nucleoside hydrolase, NSH2, is unclear. We demonstrate that NSH1 activates NSH2 in vitro and in vivo, forming a complex with almost two orders of magnitude higher catalytic efficiency for xanthosine hydrolysis than observed for NSH1 alone. Remarkably, an inactive NSH1 point mutant can activate NSH2 in vivo, fully preventing purine nucleoside accumulation in nsh1 background. Our data lead to an altered model of purine nucleotide catabolism that includes an NSH heterocomplex as a central component.

The ribokinases of Arabidopsis thaliana and Saccharomyces cerevisiae are required for ribose recycling from nucleotide catabolism, which in plants is not essential to survive prolonged dark stress.

Nucleotide catabolism in Arabidopsis thaliana and Saccharomyces cerevisiae leads to the release of ribose, which requires phosphorylation to ribose-5-phosphate mediated by ribokinase (RBSK). We aimed to characterize RBSK in plants and yeast, to quantify the contribution of plant nucleotide catabolism to the ribose pool, and to investigate whether ribose carbon contributes to dark stress survival of plants. We performed a phylogenetic analysis and determined the kinetic constants of plant-expressed Arabidopsis and yeast RBSKs. Using mass spectrometry, several metabolites were quantified in AtRBSK mutants and double mutants with genes of nucleoside catabolism. Additionally, the dark stress performance of several nucleotide metabolism mutants and rbsk was compared. The plant PfkB family of sugar kinases forms nine major clades likely representing distinct biochemical functions, one of them RBSK. Nucleotide catabolism is the dominant ribose source in plant metabolism and is highly induced by dark stress. However, rbsk cannot be discerned from the wild type in dark stress. Interestingly, the accumulation of guanosine in a guanosine deaminase mutant strongly enhances dark stress symptoms. Although nucleotide catabolism contributes to carbon mobilization upon darkness and is the dominant source of ribose, the contribution appears to be of minor importance for dark stress survival.

METABOLISM OF PURINE NUCLEOSIDES AND BASES IN SUSPENSION-CULTURED ARABIDOPSIS THALIANA CELLS

We study the metabolism of purine nucleosides and purine bases in suspension-cultured cells of the model plant Arabidopsis thaliana . [8- 14 C]Adenosine, [8- 14 C]guanosine, [8- 14 C]inosine, [8- 14 C]xanthosine, [8- 14 C]adenine, [8- 14 C]guanine, [8- 14 C]hypoxanthine and [8- 14 C]xanthine were administered to cells in the cell division phase, and the uptake and metabolic fate of these compounds were monitored for 4h. The rates of uptake of most purines were within the range 60-70 nmol/gFW. Xanthine and xanthosine were taken up more slowly. The rate of uptake was ordered as hypoxanthine > adenine > inosine > guanosine > guanine > adenosine > xanthosine> xanthine. A large amount of radioactivity from [8- 14 C]adenosine, [8- 14 C]guanosine, [8- 14 C]adenine and [8- 14 C]guanine, and a limited amount from [8- 14 C]inosine and [8- 14 C]hypoxanthine, was incorporated into nucleotides and RNA. The so-called purine salvage pathways of adenosine, guanosine, adenine, guanine, inosine and hypoxanthine are therefore functional in A. thaliana . These tracer experiments also reveal that significant amounts of these compounds were converted to xanthine, and enter the catabolic pathway via allantoin. Neither xanthosine nor xanthine is used in the synthesis of nucleotides and RNA. These compounds are entirely catabolized via allantoin and allantoic acid. Deamination of adenine and guanine rings takes place at the stage of AMP deaminase and guanosine deaminase, respectively. The pattern of purine metabolism in A. thaliana is similar to that in other plants. Adenine salvage activity estimated from the metabolism of [8- 14 C]adenine, the cellular concentration of ATP, and expression of the APT1 gene encoding adenine phosphoribosyltransferase all increased markedly at the lag phase of cell proliferation . These observations imply that the salvage pathway is important during the early stages of cell culture in A. thaliana .

Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis

Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2'-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed.

Genome-wide transcriptome profiling of region-specific vulnerability to oxidative stress in the hippocampus

Neurons in the hippocampal CA1 region are particularly sensitive to oxidative stress (OS), whereas those in CA3 are resistant. To uncover mechanisms for selective CA1 vulnerability to OS, we treated organotypic hippocampal slices with duroquinone and compared transcriptional profiles of CA1 vs CA3 cells at various intervals. Gene Ontology and Biological Pathway analyses of differentially expressed genes showed that at all time points, CA1 had higher transcriptional activity for stress/inflammatory response, transition metal transport, ferroxidase, and presynaptic signaling activity, while CA3 had higher GABA-signaling, postsynaptic, and calcium and potassium channel activity. Real-time PCR and immunoblots confirmed the transcriptome data and the induction of OS by duroquinone in both hippocampal regions. Our functional genomics approach has identified in CA1 cells molecular pathways as well as unique genes, such as guanosine deaminase, lipocalin 2, synaptotagmin 4, and latrophilin 2, whose time-dependent induction following the initiation of OS may represent attempts at neurite outgrowth, synaptic recovery, and resistance against OS.

Guanosine deaminase in human serum and tissue extracts – a reappraisal of the products

Of the human salvage enzymes that deaminate ribonucleosides, two – cytidine deaminase and adenosine deaminase – have been found particularly useful for diagnostic purposes. In humans, no enzymes are present that can directly deaminate the bases of these ribonucleosides. Indeed, the only enzyme present that can directly deaminate a base is guanine deaminase, and the diagnostic usefulness of this enzyme has been well documented. The aim of this study is to identify the origin of the ammonia formed when human sera and tissue extracts are incubated with buffered guanosine, and to clarify whether the ammonia comes from the deamination of guanosine by guanosine deaminase or is produced as a result of deamination of guanine formed as a breakdown product of guanosine by purine nucleoside phosphorylase (PNP). Apparent deamination of guanosine by guanosine deaminase in human sera and tissue extracts was found to be due to two enzymes acting in tandem when the products of the reaction were examined by HPLC. The ribose was first removed from guanosine by PNP to form guanine, which was then deaminated to xanthine by guanine deaminase

Adenosine deaminase and guanosine deaminase activities in sera of patients with viral hepatitis.

In order to investigate purin and primidin metabolism pathways in hepatitis, adenosine deaminase (ADA) and guanosine deaminase (GDA) activities in sera of patients with different types and manifestations of viral hepatitis disease (A, B, C, D, E, chronic, acute) were investigated and compared with the control group of healthy individuals. Hepatitis cases were classified with respect to their serological findings and clinics. When compared all the hepatitis cases with the controls, levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase enzymes, as well as ADA and GDA, were significantly higher than the control group (p<0.01). Levels of ADA and GDA in hepatitis cases were determined as 26.07 11.98 IU/l and 2.37 1.91 IU/l, respectively. When compared their ADA and GDA levels amongst the classified hepatitis groups, there was no difference in ADA levels amongst cases (p>0.05). However, GDA levels in hepatitis A group were closed to the controls. Increase in serum ADA activities in hepatitis forms may be dependent on and reflect the increase in phagocytic activity of macrophages and maturation of T-lymphocytes, and may be valuable in monitoring in viral hepatitis cases.

Recycling of α- d-ribose 1-phosphate for nucleoside interconversion

Mobilization of the ribose moiety and of the amino group of guanosine may be realized in rat liver extract by the concerted action of purine nucleoside phosphorylase and guanase. Ribose 1-phosphate formed from guanosine through the action of purine nucleoside phosphorylase acts as ribose donor in the synthesis of xanthosine catalyzed by the same enzyme. The presence of guanase, which irreversibly converts guanine to xanthine, affects the overall process of guanosine transformation. As a result of this purine pathway, guanosine is converted into xanthosine, thus overcoming the lack of guanosine deaminase in mammals. Furthermore, in rat liver extract the activated ribose moiety stemming from the catabolism of purine nucleosides can be transferred to uracil and, in the presence of ATP, used for the synthesis of pyrimidine nucleotides; therefore, purine nucleosides can act as ribose donors for the salvage of pyrimidine bases.

Guanosine Deaminase and Guanine Deaminase from Tea Leaves

Guanosine deaminase and guanine deaminase were partially purified from tea leaves. The optimum activity of guanosine deaminase was observed at pH 7.5 and that of guanine deaminase was at pH 7.0–7.5 and 8.5. Guanosine deaminase was an unstable enzyme. The activities of these deaminases were significantly inhibited by heavy metals. Molecular weights of guanosine deaminase and guanine deaminase as measured by gel filtration were about 18,000 and 54,000, respectively. The Km for the respective substrates, guanosine and guanine, were 9.5 μm and 41.7 μm. Guanosine deaminase was considered to catalyze the deamination of 2′-deoxyguanosine besides guanosine. It is suggested that guanosine deaminase as well as guanine deaminase in tea leaves not only acts on the catabolic pathway, but also is involved in the biosynthesis of caffeine from guanosine or guanine nucleotides.

The measurement of nucleoside deaminases by high performance liquid chromatography and their use in clinical chemistry

The measurement of the nucleoside deaminases--cytidine deaminase, guanosine deaminase and adenosine deaminase--by reversed phase high performance liquid chromatography is reviewed. The clinical value of assaying the enzyme activity is discussed for each of these enzymes. Both cytidine deaminase and adenosine deaminase measurements have proven clinical value, although the use of the assay of cytidine deaminase in the diagnosis of pre-eclampsia is probably not helpful.

The estimation of serum guanosine deaminase activity in liver disease.

An assay for the estimation of guanosine deaminase is described. The method employs guanosine as substrate and after incubation of serum and substrate at 22 degrees C for 18 h the ammonia liberated is estimated using the Berthelot reaction. Absorbance is measured as 625 nm and the catalytic activity read from a standard curve obtained using ammonia standards. The method provides reproducible measurements of serum guanosine deaminase. The results obtained using 'normal' sera have been used to calculate the 'normal range' for the enzyme in serum. Preliminary results suggest that guanosine deaminase is increased in hepatitis and in patients with liver metastases but normal in all other liver diseases including cirrhosis and obstructive jaundice.

Selective Inhibition of Xanthine Oxidase by 3-Hydroxy-1-nitrophenyl-1H-pyrazolo[4,3-c]pyridines

Some 3-hydroxy-1-nitrophenyl-1H-pyrazolo[4,3-c]-pyridines were synthesized and characterized. The ability of the compounds to inhibit purine-catabolizing enzymes was evaluated. These pyrazolopyridines were found to exhibit selective inhibitory effects on xanthine oxidase, while adenosine deaminase, guanosine deaminase, and guanine deaminase were unaffected. 3-Hydroxy-1-(2',4'-dinitrophenyl)-1H-pyrazolo[4,3-c]pyridine was the most potent inhibitor of xanthine oxidase.

細菌によるグアノシンの分解について(第1報)

It is well known that adenine derivatives are deaminated by adenase, adenosine deaminase and adenylic acid deaminase. However, only guanase has been known for guanine derivatives as the deaminating enzymes. Degradation of guanosine by bacterial enzymes has been known to proceed by a combination of nucleoside phosphorylase or nucleosidase and guanase yielding xanthine. The authors isolated about 400 bacteria from soil and found that a strain No. 149 seemed to produce a new enzyme, guanosine deaminase which directly deaminates guanosine producing xanthosine. Furthermore, the morphological, cultural and physiological properties of this strain were investigated. From these results, the bacterium was classified as Pseudomonas convexa CHESTER.