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DNA damage and oxidative stress play a critical role in photoageing. Seborrhoeic keratosis (SK) affects sunlight-exposed sites in aged individuals. This study examined the mechanism of photoageing in SK. The guanine deaminase gene, which is involved in purine metabolism, was upregulated with uric acid levels and p21 in SK. Guanine deaminase was detectable in keratinocytes. Repeated exposure to ultraviolet (UV) increased levels of guanine deaminase, together with DNA damage, such as γ-H2AX and cyclobutane pyrimidine dimer formation, generation of reactive oxygen species, and keratinocyte senescence, which were reversed by guanine deaminase knockdown. However, guanine deaminase overexpression and H2O2 formed γ-H2AX, but not cyclobutane pyrimidine dimer. Loss-of-function guanine deaminase mutants reduced the metabolic end-product uric acid, which was increased by exposure to exogenous xanthine. Repeated exposure to UV increased levels of uric acid. Exogenous uric acid increased cellular senescence, reactive oxygen species, and γ-H2AX, similar to guanine deaminase. Overall, guanine deaminase upregulation increased UV-induced keratinocyte senescence in SK, via uric acid mediated by reactive oxygen species followed by DNA damage.

As endogenous biomolecules, guanine, guanine-based nucleosides, and nucleotides are essential for cellular DNA/RNA synthesis, energy metabolism, and signal transduction. However, these biomolecules have been found to have a cell-specific antiproliferation effect at higher concentrations, and the mechanism is unclear. In this study, we demonstrate that guanine deaminase (GDA) is a major factor in determining the cell-type selectivity to the antiproliferation effect of guanine-based biomolecules. GDA catalyzes the deamination of guanine to xanthine, which is an essential part of the guanine degradation pathway. GDA deficient cells could not efficiently remove the excess guanine-based biomolecules. These excess molecules disturb the metabolism of adenine-, cytosine-, and thymine-based nucleotides; subsequently inhibit the DNA synthesis and cell growth; and eventually result in the apoptosis/death of GDA deficient cells. The inhibition of DNA synthesis could be relieved by simultaneous addition of adenine- and cytosine-based nucleosides, and the inhibited DNA synthesis could be restarted by post addition of them, which subsequently reduces the antiproliferation effect of guanine-based biomolecules or even totally restores the cell proliferation. These results provide important information for the development of guanine-based drugs or guanine-rich oligonucleotide drugs, as well as for the safety evaluation of food with a high level of guanine-based compounds.

Guanine deaminase (GDA; cypin) is an important metalloenzyme that processes the first step in purine catabolism, converting guanine to xanthine by hydrolytic deamination. In higher eukaryotes, GDA also plays an important role in the development of neuronal morphology by regulating dendritic arborization. In addition to its role in the maturing brain, GDA is thought to be involved in proper liver function since increased levels of GDA activity have been correlated with liver disease and transplant rejection. Although mammalian GDA is an attractive and potential drug target for treatment of both liver diseases and cognitive disorders, prospective novel inhibitors and/or activators of this enzyme have not been actively pursued. In this study, we employed the combination of protein structure analysis and experimental kinetic studies to seek novel potential ligands for human guanine deaminase. Using virtual screening and biochemical analysis, we identified common small molecule compounds that demonstrate a higher binding affinity to GDA than does guanine. In vitro analysis demonstrates that these compounds inhibit guanine deamination, and more surprisingly, affect GDA (cypin)-mediated microtubule assembly. The results in this study provide evidence that an in silico drug discovery strategy coupled with in vitro validation assays can be successfully implemented to discover compounds that may possess therapeutic value for the treatment of diseases and disorders where GDA activity is abnormal., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

An enzyme from Pseudomonas aeruginosa, Pa0142 (gi|9945972), that is able to catalyze the deamination of 8-oxoguanine (8-oxoG) to uric acid has been identified for the first time. 8-Oxoguanine is formed by the oxidation of guanine residues within DNA by reactive oxygen species, and this lesion results in G:C to T:A transversions. The value of k(cat)/K(m) for the deamination of 8-oxoG by Pa0142 at pH 8.0 and 30 degrees C is 2.0 x 10(4) M(-1) s(-1). This enzyme can also catalyze the deamination of isocystosine and guanine at rates that are approximately an order of magnitude lower. The three-dimensional structure of a homologous enzyme (gi|44264246) from the Sargasso Sea has been determined by X-ray diffraction methods to a resolution of 2.2 A (PDB entry). The enzyme folds as a (beta/alpha)(8) barrel and is a member of the amidohydrolase superfamily with a single zinc in the active site. This enzyme catalyzes the deamination of 8-oxoG with a k(cat)/K(m) value of 2.7 x 10(5) M(-1) s(-1). Computational docking of potential high-energy intermediates for the deamination reaction to the X-ray crystal structure suggests that active-site binding of 8-oxoG is facilitated by hydrogen-bond interactions from a conserved glutamine that follows beta-strand 1 with the carbonyl group at C6, a conserved tyrosine that follows beta-strand 2 with N7, and a conserved cysteine residue that follows beta-strand 4 with the carbonyl group at C8. A bioinformatic analysis of available protein sequences suggests that approximately 200 other bacteria possess an enzyme capable of catalyzing the deamination of 8-oxoG.

Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in GDEase activity, a collection of mutant strains was screened for strains unable to use guanine as a nitrogen source. The strain BFA1819 (yknA) showed the expected phenotype and no GDEase activity could be detected in this strain. A new name for yknA, namely gde, is proposed. The gde gene encodes a 156 amino acid polypeptide and was preceded by a promoter sequence that is recognized by the sigma(A) form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular mass inducer molecule. The level of GDEase was found to be subjected to global nitrogen control exerted by the GlnA/TnrA-dependent signalling pathway. The two regulatory proteins of this pathway, TnrA and GlnR, indirectly and positively affected gde expression. This is the first instance of a gene whose expression is positively regulated by GlnR. The GDEase amino acid sequence shows no homology with the mammalian enzyme. In agreement with this are the different physiological roles for the two enzymes.

The preservation of purine ring as purine bases appears to be a common feature of camel liver. Hepatic guanine appears to be actively converted into GMP in the camel rather than further degraded. The limiting step of guanine degradation appears to be the lack of hepatic guanase activity. Higher purine bases over uric acid ratios were found in camel urine with respect to those of zebu.