Cloning and characterization of human guanine deaminase - Purification andpartial amino acid sequence of the mouse protein

Mouse erythrocyte guanine deaminase has been purified to homogeneity, The n ative enzyme was dimeric, being comprised of two identical subunits of appr oximately 50,000 Da, The protein sequence was obtained from five cyanogen b romide cleavage products giving sequences ranging from 12 to 25 amino acids in length and corresponding to 99 residues. Basic Local Alignment Search T ool (BLAST) analysis of expressed sequence databases enabled the retrieval of a human expressed sequence tag cDNA clone highly homologous to one of th e mouse peptide sequences. The presumed coding region of this clone was use d to screen a human kidney cDNA library and secondarily to polymerase chain reaction-amplify the full-length coding sequence of the human brain cDNA c orresponding to an open reading frame of 1365 nucleotides and encoding a pr otein of 51,040 Da, Comparison of the mouse peptide sequences with the infe rred human protein sequence revealed 88 of 99 residues to be identical. The human coding sequence of the putative enzyme was subcloned into the bacter ial expression vector pMAL-c2, expressed, purified, and characterized as ha ving guanine deaminase activity with a K-m for guanine of 9.5 +/- 1.7 mu M. The protein shares a 9-residue motif with other aminohydrolases and amidoh ydrolases (PGX[VI]DXH[TVI]H) that has been shown to be ligated with heavy m etal ions, commonly zinc, The purified recombinant guanine deaminase was fo und to contain approximately 1 atom of zinc per 51-kDa monomer.