This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fumagillin is among the most effective compounds for treating microsporidiosis. The putative target of this compound is methionine aminopeptidase 2 (MetAP2). This report describes the characterization and phylogenetic analysis of the deduced amino acid sequences of MetAP-2 enzymes from microsporidian species and genotypes of the genus Encephalitozoon. Fragments of DNA encoding 318 to 335 amino acid residues of the MetAP-2 genes were isolated from genomic DNA prepared from cultured spores of E. hellem, E. intestinalis, and E. cuniculi genotypes I, II and III. Sequence comparisons of the deduced amino acid residues indicated that the microsporidian sequences are MetAP-2-like rather than MetAP-1-like. Alignments demonstrated that the new Encephalitozoon sequences included sequences and structures conserved in eukaryotic MetAP-2s, including the five conserved, active site residues, Asp, Asp, His, Glu and Glu, considered to be critical for catalysis and for coordinating the cation (eg. Cobalt) co-factor, and included residues known to interact with the antibiotic, fumagillin. The primary structure of the Encephalitozoon MetAP-2s, however, showed some dissimilarity with human and yeast MetAP-2s, including the absence of the NH2-terminal polylysine tract. Phylogenetic comparison of these Encephalitozoon MetAP-2s with orthologues from related species and from other informative taxa confirmed that the MetAP-2s of these Encephalitozoon species and strains are closely related to each other and cluster with MetAP-2s. The Encephalitozoon MetAP2s have been expressed and protease activity was inhibited by an irreversible covalent-binding inhibitor, the fumagillin analogue, TNP-470, as well as by a non-covalent reversible inhibitor, bestatin. Studies are underway to develop a high throughput screening assay for inhibitors of Encephalitozoon and Enterocytozoon MetAP2s that are as effective, but less toxic, than fumagillin.
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