Gene PH0095 from Pyrococcus horikoshii codes for a hypoxanthine, guanine, xanthine phosphoribosyltransferase
Dantas, D.S.; Pereira, G.A.G.; Medrano, F.J.
Centro de Biologia Molecular Estrutural, LNLS, Campinas, SP
Laboratorio de Genômica e Expressão UNICAMP, Campinas, SP
Purine phosphoribosyltransferases are involved in the salvage of purine bases. They catalyse the reversible transfer, dependent of magnesium, of a phosphoribosyl group from 5-phospho-a-D-ribosyl 1-pyrophosphate (PRPP) to a purine base (hypoxanthine, guanine or xanthine) releasing pyrophosphate, resulting in the correnponding nucleotide monophosphate (IMP, GMP or XMP). HPRT from P. horikoshii shows significant differences in key residues present in three of the four catalytic loops. Thermal stability is an important aspect of proteins, not completely understood. Here, with the study of this protein from a hyperthermophylic microorganism we would like to get some insights into the sequence rearrangements that a protein should go through in order to gain the necessary stability to perform its activity at high temperature.
We have cloned the gene coding for the HPRT from P. horikoshii in the pET15b expression vector, and expressed in Escherichia coli BL21 StarTM (DE3) (Invitrogen). The protein was purified in two steps: heating at 75ºC for 1 hour followed by centrifugation to eliminate all the precipitated material and a second step of immobilized metal affinity chromatography.
We have carried out steady-state kinetic assays in order to discover the specificity of this protein. These experiments show that the protein is able to catalyze the formation of IMP, GMP and XMP. Thus, this protein is a hypoxanthine, guanine, xanthine phosphoribosyltransferase. The reaction is more efficient with hypoxanthine, followed by guanine and xanthine, and there is a great dependence of the catalytic constant with the temperature, being more efficient at higher temperatures.
In summary, we have cloned, expressed and purified an extremely stable protein containing a flexible region. Also, we have demonstrated that this protein is able to salvage hypoxanthine, guanine and xanthine. Further studies will give us some insights on the effect of sequence rearrangements on the final structure of the protein.
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