| The infectious stage of the parasitic protozoan Entamoeba histolytica is the environmentally resistant cyst. The major component that forms the cyst wall of Entamoeba invadens, a model for the human-infectious parasite Entamoeba histolytica, is chitin, which is a homopolymer of β-1, 4-linked N-acetyl-glucosamine. The first step in the hexosamine pathway leading to the formation of the chitinaceous cyst wall is the interconversion between fructose 6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). The enzymes that catalyze this reaction in the anabolic and catabolic direction are GlcN6P synthase and GlcN6P deaminase (also known as GlcN6P isomerase or Gpi), respectively, two enzymes that are markedly different at the primary structure level. Although primarily a deaminase converting GlcN6P to Fru6P and ammonia, it has been suggested that Gpi may run in the aminating direction in some organisms. The genome of Entamoeba histolytica contains a gene that encodes a protein with significant homology to prokaryotic and some eukaryotic gpis. Unlike any other characterized glucosamine phosphate isomerase, however, the amino acid sequence of the amoebic enzyme predicts the presence of an additional domain with homology to the mammalian de-N-acetylase, PIG-L. The second step in the hexosamine pathway is the acetylation of GlcN6P; therefore, the presence of this additional domain suggests that the amoebic Gpi might act as a bifunctional enzyme, carrying out the first two steps of this pathway as a single unit. As part of the characterization of this protein, we examined the evolutionary origin of this long form of Gpi and found that the gene was likely introduced into the ancestors of Amoebozoa, Ciliophora and Heterolobosea as a result of at least three independent lateral gene transfer events. To further characterize this enzyme, we cloned the E. histolytica Gpi. The enzyme appears to be a hexamer with a molecular weight of approximately 459kDa. We detected enzyme activity in the aminating, deaminating and acetylating directions. The kinetic parameters for the above reactions have been determined under hyperbolic conditions. |
