Identification, expression, and characterization of Escherichia coli guanine deaminase

Using the human cDNA sequence corresponding to guanine deaminase, the Esche richia call genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown func tion was identified as having 36% identity to the human protein, The putati ve gene was amplified, subcloned into the pMAL-c2 vector, expressed, purifi ed, and characterized enzymatically. The 50.2-kDa protein catalyzed the con version of guanine to xanthine, having a K-m of 15 mu M with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. c oli guanine deaminase locus is 3' from an open reading frame which shows ho mology to a bacterial purine base permease.