CLONING, EXPRESSION AND CHARACTERIZATION OF AN UNUSUAL GUANINE PHOSPHORIBOSYLTRANSFERASE FROM GIARDIA-LAMBLIA

Giardia lamblia is one of the most ancient eukaryotes identified to da te. It lacks de novo purine biosynthesis and is thought to rely solely on the functions of two salvage enzymes, adenine and guanine phosphor ibosyltransferases (APRTase and GPRTase). We have cloned the gene enco ding the G. lamblia GPRTase by complementation of the E. coli strain S O609 (Delta gpt-pro-lac, thi, hpt, pup, pur H,J, strA) with a genomic library consisting of Sau3AI-digested G. lamblia DNA inserted into the Bluescript(TM) vector. Transformed SO609 colonies grew on minimal med ium supplemented with guanine at a frequency of 3.3 x 10(-5) ampicilli n-resistant colonies, but were unable to salvage hypoxanthine or xanth ine, as predicted from previous studies of the native G. lamblia GPRTa se. The sequence analysis of cloned DNA fragments reveals an open read ing frame of 690 bp, encoding a protein of 26.3 kDa with an estimated pi of 6.83, in agreement with the reported subunit molecular weight of the native G. lamblia GPRTase. The deduced protein has less than 20% sequence identity to the human and other known HGPRTases, and features several significant changes in the primary sequence of the putative a ctive sites of the enzyme, which may reflect the stringent substrate s pecificity of GPRTase. The recombinant GPRTase was expressed in E. col i and purified to >95% homogeneity. Kinetic studies of the recombinant enzyme showed an apparent K-m of 74 mu M for guanine. Hypoxanthine as an alternate purine substrate was used only when present in millimola r amounts, and xanthine was not utilized at all. This Giardia enzyme i s thus a highly unique purine PRTase without a known parallel in any o ther living organisms.