Abstract
Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis. Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1. The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46). Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell. Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T. cruzi cells. Southern and Western analysis suggests that PEP mutase is unique to T. cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.
Highlights
Phosphonates are widely distributed in nature as components of lipids, oligosaccharides, and proteins as well as microbial secondary metabolites with antibacterial, antimalarial, and herbicidal activities [1, 2]
The left panel protein blot was probed with anti-serum to T. cruzi PEP mutase (TcPEPM); the right panel was probed with antibody to T. cruzi trypanothione synthetase (TcTryS)
The partial sequence of the T. cruzi PEP mutase gene (PEPM) was amplified by PCR and used as a hybridization probe for a Southern blot analysis of genomic DNA digested with a range of restriction enzymes
Summary
Phosphonates are widely distributed in nature as components of lipids, oligosaccharides, and proteins as well as microbial secondary metabolites with antibacterial (e.g. fosfomycin), antimalarial (fosmidomycin), and herbicidal (e.g. bialaphos) activities [1, 2]. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis.
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