A novel Escherichia coli strain allows functional analysis of guanylate kinase drug resistance and sensitivity.
Guanylate kinase is a critical enzyme in the biosynthesis of guanosine 5'-triphosphate (GTP) and dGTP and is responsible for the phosphorylation of guanosine 5'-monophosphate (GMP) and dGMP to guanosine 5'-diphosphate (GDP) and dGDP, respectively. As with many nucleotide-metabolizing enzymes, guanylate kinase is involved in antimicrobial and antineoplastic drug activation. This is due to the structural similarities of such agents with nucleobases or nucleosides that are acted upon by endogenous enzymes. Despite the involvement of guanylate kinase in 6-thioguanine, mercaptopurine, and abasic guanosine analog (e.g., ganciclovir) activation, studies have only recently focused on the molecular basis of the structure to function relationship of a mammalian guanylate kinase. As a means to evaluate the details of amino acid side chain involvement in substrate interaction, we have constructed a conditional guanylate-kinase-deficient Escherichia coli strain that requires the presence of a functional, plasmid-borne guanylate kinase for growth under selective conditions. Positive genetic selection provides a rapid mechanism to identify not only functional guanylate kinase mutants but also those that result in drug resistance. This novel strain will be beneficial to assess the role of specific amino acids of guanylate kinase in structure, function, drug activation, and drug resistance.
Stolworthy
Tiffany S
TS
Department of Pharmaceutical Sciences, P.O. Box 646534, Washington State University, Pullman, WA 99164-6534, USA.
Krabbenhoft
Elizabeth
E
Black
Margaret E
ME
D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D020868:Gene Silencing; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051528:Guanylate Kinases; D009154:Mutation; D009703:Nucleoside-Phosphate Kinase / Q000172:deficiency / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D013866:Thioguanine / Q000494:pharmacology
D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases; D013866:Thioguanine