Cloning, Characterization, and Modeling of Mouse and Human Guanylate Kinases

Abstract

Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways. Despite its involvement in antiviral drug activation in humans and in mouse model systems and as a target for chemotherapy, the human and mouse primary structures have never been elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinase were isolated from mouse B-cell lymphoma and human peripheral blood lymphocyte cDNA libraries. Multiple tissue Northern blots demonstrated an mRNA species of approximately 1 kilobase for both mice and humans in all tissue types examined. The mouse cDNA is predicted to encode a 198-amino acid protein with a molecular mass of 21,904 daltons. The human cDNA is predicted to encode a 197-amino acid protein with a molecular mass of 21,696 daltons. These proteins share 88% sequence identity with each other and 52-54% identity with the yeast guanylate kinase. Molecular modeling using the yeast diffraction coordinates indicates a high degree of conservation within the active site and maintenance of the overall structural integrity, despite a lack of similarity along the periphery of the enzyme.Guanylate kinase (GMK, 1 ATP:GMP phosphotransferase; EC 2.7.4.8) catalyzes the reaction (d)GMP ϩ ATP 3 (d)GDP ϩ ADP where (d)GMP indicates GMP or dGMP. In addition to being a critical enzyme in the biosynthesis of GTP and dGTP, guanylate kinase functions in the recovery of cGMP (cGMP 3 GMP 3 GDP 3 GTP 3 cGMP) and is, therefore, thought to regulate the supply of guanine nucleotides to signal transduction pathway components (1, 2).As with other enzymes involved in nucleotide metabolism, guanylate kinase is a target for cancer chemotherapy and is inhibited by the potent antitumor drug, 6-thioguanine (3-5). Guanylate kinase activity is also required for the potentiation of antiviral drug activity in virus-infected cells. Activation of the anti-herpes guanosine nucleoside analogs, acyclovir and ganciclovir, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase (6, 7).Although guanylate kinase is a key enzyme for cancer chemotherapy and antiviral drug activation in humans, the gene encoding this important enzyme has not been described. In this study, we describe the isolation, expression, and functional analysis of both human and mouse guanylate kinases in an effort to aid improvements in antiviral and tumor chemotherapeutic approaches through molecular modeling and rational drug design. EXPERIMENTAL PROCEDURESMaterials-Restriction enzymes, deoxynucleoside triphosphates, and random-primed DNA labeling kits were purchased from Boehringer Mannheim. Reagents for enzyme assays were purchased from Sigma.[␣-32 P]dCTP (3000 Ci/mmol) and 14 C-labeled protein molecular weight markers were purchased from Amersham Corp. 35S Express protein labeling mix was purchased from DuPont NEN. Biotinylated thrombin, pET23d vector, and strepavidin beads were obtained from Novagen (Madison, WI). Multiple tissue ...

The contribution of CMP kinase to the efficiency of DNA repair

“…These RNR substrates, NDPs-ADP, GDP, CDP and UDP, are generated by AMP kinase, GMP kinase and UMP/CMP kinase (CMPK) from corresponding NMPs, respectively. [8][9][10] Human CMPK has been identified ( EC 2.7.4.14) and biochemically characterized.…”

A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing

“…[15][16][17][18] From an enzyme kinetic perspective, the high K m of GMK for GCV-MP (K m ¼ 42-54 mM) compared to the K m for GMP (B25 mM) supports the notion that low endogenous GMK activity may limit production of the pharmacologically active form of GCV. 15,19 As a means to overcome the bottleneck of prodrug activation at the mono-to diphosphate step, we combined both the critical HSVTK and GMK activities together in a single fusion or chimeric protein and assessed the ability of pathway engineering to reduce to the level of GCV required for effective cell killing.…”

“…10,19 The resulting plasmid, designated pET23d:mgmk/TK, was sequenced to confirm in-frame fusion of the two genes. As a result of the cloning sites used, the first nine amino acids of HSVTK and the last two amino acids of MGMK were deleted.…”

“…Overexpression of TK, Mgmk and mgmk/TK from pET vector constructs was as previously reported. 11,19 Mgmk was expressed from pETHT which fuses a histidine tag to MGMK and the enzyme purified using nickel affinity chromatography. 19 Purification of TK and MGMK/TK was performed by affinity chromatography using CH-Sepharose 4B-coupled p-aminophenylthymidine 3'-phosphate resin as previously described.…”

A Mutation in the Essential Gene gmk (Encoding Guanlyate Kinase) Generates a Requirement for Adenine at Low Temperature in Salmonella enterica

“…In addition, the biomedical role of human Gmk in the activation of antiviral agents has attracted significant attention (2, 13, 14). As a result, a large amount of information has amassed about the biochemistry, genetics, and regulation of these enzymes for a variety of organisms (2,5,6,8,12,13,15,17,20,23) and a high-resolution structure for the Saccharomyces cerevisiae enzyme (GUK1) is available (21). Most guanylate kinases have significant sequence similarity, and the conserved motifs allow the prediction of substrate binding sites.…”