Cloning, expression and characterization of an unusual guanine phosphoribosyltransferase from Giardia lamblia.
Giardia lamblia is one of the most ancient eukaryotes identified to date. It lacks de novo purine biosynthesis and is thought to rely solely on the functions of two salvage enzymes, adenine and guanine phosphoribosyltransferases (APRTase and GPRTase). We have cloned the gene encoding the G. lamblia GPRTase by complementation of the E. coli strain Sø609 (delta gpt-pro-lac, thi, hpt, pup, purH,J, strA) with a genomic library consisting of Sau3AI-digested G. lamblia DNA inserted into the Bluescript vector. Transformed Sø609 colonies grew on minimal medium supplemented with guanine at a frequency of 3.3 x 10(-5) ampicillin-resistant colonies, but were unable to salvage hypoxanthine or xanthine, as predicted from previous studies of the native G. lamblia GPRTase. The sequence analysis of cloned DNA fragments reveals an open reading 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 active sites of the enzyme, which may reflect the stringent substrate specificity of GPRTase. The recombinant GPRTase was expressed in E. coli and purified to > 95% homogeneity. Kinetic studies of the recombinant enzyme showed an apparent K(m) of 74 microM for guanine. Hypoxanthine as an alternate purine substrate was used only when present in millimolar amounts, and xanthine was not utilized at all. This Giardia enzyme is thus a highly unique purine PRTase without a known parallel in any other living organisms.
Sommer
J M
JM
University of California, Department of Pharmaceutical Chemistry, San Francisco 94143-0446, USA. jsommer@cgl.ucsf.edu
Ma
H
H
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase