Cloning and Characterization of Human Guanine Deaminase

Abstract

Mouse erythrocyte guanine deaminase has been purified to homogeneity. The native enzyme was dimeric, being comprised of two identical subunits of approximately 50,000 Da. The protein sequence was obtained from five cyanogen bromide cleavage products giving sequences ranging from 12 to 25 amino acids in length and corresponding to 99 residues. Basic Local Alignment Search Tool (BLAST) analysis of expressed sequence databases enabled the retrieval of a human expressed sequence tag cDNA clone highly homologous to one of the mouse peptide sequences. The presumed coding region of this clone was used to screen a human kidney cDNA library and secondarily to polymerase chain reaction-amplify the full-length coding sequence of the human brain cDNA corresponding to an open reading frame of 1365 nucleotides and encoding a protein of 51,040 Da. Comparison of the mouse peptide sequences with the inferred human protein sequence revealed 88 of 99 residues to be identical. The human coding sequence of the putative enzyme was subcloned into the bacterial expression vector pMAL-c2, expressed, purified, and characterized as having guanine deaminase activity with a K m for guanine of 9.5 ؎ 1.7 M. The protein shares a 9-residue motif with other aminohydrolases and amidohydrolases (PGX[VI]DXH[TVI]H) that has been shown to be ligated with heavy metal ions, commonly zinc. The purified recombinant guanine deaminase was found to contain approximately 1 atom of zinc per 51-kDa monomer.Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine. By producing xanthine and ammonia, this reaction irreversibly eliminates the guanine base from further reutilization as a guanylate nucleotide in mammals. The product xanthine is a substrate for xanthine oxidase in the production of uric acid. Although it is an enzyme of purine catabolism, guanine deaminase is not ubiquitously expressed and exhibits a general absence in lymphoid tissues and variable expression elsewhere (1, 2). The highest levels of expression were found in the proximal section of the small intestine of the mouse (3). There are greater than 50-fold differences in guanine deaminase among different regions of the mouse brain; the cerebral cortex and amygdala have the highest activity (4), whereas there is essentially no activity in the cerebellum of the mouse or cat (4, 5). There are greater than 10-fold increases in the level of expression of guanine deaminase in the liver, kidney, and brain during the 40-day postnatal development of the rat (6), and alterations in embryonic expression have also been characterized (7). In the adult mouse, fractional increases in brain and liver enzyme activity occur in response to intraperitoneal administration of a bolus of guanine (8). The tissue-specific expression and the developmental and induced changes in expression suggest a potential role for guanine deaminase in the regulation of the guanine nucleotide pool. Cellular GTP has an important role not only in specific enzyme reactions and protein synth...

Barbiturase, a Novel Zinc-containing Amidohydrolase Involved in Oxidative Pyrimidine Metabolism

“…The dihydro-orotase from hamster was suggested to contain two atoms of zinc per mol of enzyme (26), whereas recently the E. coli enzyme was revealed to have four atom of zinc per mol of enzyme by three-dimensional structure analysis (41). A recently purified and cloned human guanine deaminase (which also possesses the metal binding motif DXHXH) was determined to contain two atoms of zinc per mol of enzyme (42). The zinc-binding site of barbiturase is located at amino acids 320 -324 (DVHWH, Fig.…”

A Novel NE-dlg/SAP102-associated Protein, p51-nedasin, Related to the Amidohydrolase Superfamily, Interferes with the Association between NE-dlg/SAP102 and N-Methyl-d-aspartate Receptor

“…These proteins have a histidine-aspartic acid signature which is required for metal binding (27) and are known to catalyze hydrolytic reactions with nitrogen heterocyclic ring substrates (29). During the preparation of this manu- script, cDNA encoding a human protein having guanine aminohydrolase (GAH) activity was reported (28), and its deduced amino acid sequence is identical to nedasin. GAH catalyzes the hydrolytic deamination of guanine, yielding xanthine and ammonium, and is considered to be involved in a major pathway for producing uric acid (30).…”

“…Notably, the N-terminal HXH motif of the signature pattern and the C-terminally conserved block of the amidohydrolase superfamily are highly conserved in nedasin. A recent report (28) shows that the protein that harbors a guanine aminohydrolase activity appears to have an identical sequence to nedasin, suggesting that nedasin may be involved in intracellular guanine metabolism.…”

Catalytic Mechanism of Guanine Deaminase: An ONIOM and Molecular Dynamics Study

“…1,2 Two families of GDs have evolved in nature, one with ∼160 amino residues such as Bacillus subtilis GD (bGD) 1 and the other with >400 residues such as Escherichia coli 3 and mammalian GDs. [4][5][6] In mammals, the GD gene expression is tissue-specific and development-dependent. 4,5,7,8 Because of its near absence in normal human serum, erythrocytes, and lymphoid cells, the GD activity is a specific and sensitive index for the diagnosis of liver diseases.…”

“…[4][5][6] In mammals, the GD gene expression is tissue-specific and development-dependent. 4,5,7,8 Because of its near absence in normal human serum, erythrocytes, and lymphoid cells, the GD activity is a specific and sensitive index for the diagnosis of liver diseases. 9,10 The crystal structure of bGD has been determined at 1.17 Å resolution.…”