Differential inhibitory effects of GMP‐2′,3′‐dialdehyde on human and schistosomal hypoxanthine–guanine phosphoribosyltransferases

Abstract

The hypoxanthine–guanine phosphoribosyltransferase (HGPRTase) of human and the parasitic trematode, Schistosoma mansoni, were expressed at high levels in transformed Escherichia coli in their native forms. Guanosine 2′,3′‐dialdehyde 5′‐phosphate (ox‐GMP) was shown to bind irreversibly to both enzymes in a time‐dependent manner. This binding was stabilized by sodium borohydride reduction, suggesting that a Schiff's base is formed between the dialdehyde groups of ox‐GMP and the amino group of a lysine residue in the enzymes. This linkage formation applies also to inosine 2′,3′‐dialdehyde 5′‐phosphate but not to adenosine 2′,3′‐dialdehyde 5′‐phosphate. GMP was found to be protective against ox‐GMP inactivation and [3H]ox‐GMP labeling of both HGPRTases. 5‐Phosphoribosyl‐1‐diphosphate (PRibPP) also protects human HGPRTase against the ox‐GMP inactivation and [3H]ox‐GMP labeling but provides virtually no protection against the ox‐GMP inactivation and labeling of the schistosomal enzyme, even though PRibPP binds to the latter with a threefold higher affinity. These results imply that PRibPP and ox‐GMP compete with each other for binding to the human HGPRTase but not for binding to the schistosomal enzyme. This discrepancy could be exploited for the purpose of designing selective inhibitors of the schistosomal HGPRTase. Guanosine 2′,3′‐dialdehyde (ox‐guanosine) is nearly as active as ox‐GMP in inhibiting schistosomal HGPRTase but much less potent in inhibiting human HGPRTase, suggesting that ox‐guanosine and ox‐GMP may bind equally well to the parasite enzyme. PRibPP can protect human but not schistosomal HGPRTase against the inactivation by ox‐guanosine. Therefore, ox‐GMP and ox‐guanosine must be forming Schiff's bases with the same amino acid residues in each of the two HGPRTases.

Virtual Screening of Combinatorial Libraries across a Gene Family: in Search of Inhibitors of Giardia lamblia Guanine Phosphoribosyltransferase

“…Isolation (5,14) and assays (34) of recombinant G. lamblia GPRT and human HGPRT from transformed Escherichia coli were performed as described previously. The substrates were present at the following concentrations: 20 M guanine (K M ϭ 16.4 M) and 1 mM PRPP (K M ϭ 25.6 M).…”

Converting the Guanine Phosphoribosyltransferase from Giardia lamblia to a Hypoxanthine-guanine Phosphoribosyltransferase

“…The purified enzyme samples were stored at Ϫ80°C with no detectable loss of activity after 4 months. The recombinant human HGPRTase was purified from E. coli S606, transformed with pBAcprt expression vector, by a previously described procedure (18).…”

“…Following the polymerase chain reaction, mutant plasmid was transformed into E. coli S606. Plasmid DNA isolated from the transformants were sequenced for verification, and the recombinant mutant protein was then purified from the transformed E. coli lysate using the same procedure as for the wild-type enzyme purification (16,18). The stability of each mutant enzyme activity was tested in repeated assays.…”

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets

“…Studies on the biological, chemical and pharmacological aspects of pyrimidine metabolism in various organisms, from prokaryotes to mammalian systems, are numerous and have been discussed in several reviews by O’Donovan and Neuhard (1970), Henderson and Paterson (1973), Levine et al (1974), Jaffe (1975), Hurst (1980), Jones (1980), Kensler and Cooney (1981), Munch-Peterson (1983), Hammond and Gutteriddge (1984), Hassan and Coombs (1988), Evans and Guy (2004), Hyde (2007), Garavito et al (2015), and Krungkrai and Krungkrai (2016). However, in contrast to the relatively extensive work on purine metabolism in schistosomes (Senft et al, 1972, 1973a and 1973b; Stegman et al, 1973; Crabtree and Senft, 1974; Miech et al, 1975; Levy and Read, 1975a and 1975b; Senft and Crabtree, 1983; el Kouni et al, 1983a, 1985, 1987 and 1989; Dovey, et al, 1984 and 1985; el Kouni and Cha, 1987; Baer et al, 1988; el Kouni, 1991; Craig III et al, 1991; Yuan et al, 1993; Kanaaneh et al, 1994: Kanaani et al, 1995 and 1997; Foulk et al, 2002; Pereira et al, 2003, 2005, 2010a and 2010b; da Silveira et al, 2004; Yang et al, 2007; Castilho et al, 2010; D’Muniz-Pereira et al, 2011; Postigo et al, 2010; Marques-Ide et al, 2012; de Moraes et al, 2013; Romanello et al, 2013 and 2017; Saverese and el Kouni, 2014; Torini et al, 2016; Zeraik et al, 2017), little information is available on pyrimidine metabolism in this parasite.…”