The two Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers

Abstract

Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I·HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I-HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.