Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase

Alkyl dihydroxyacetone-phosphate synthase is the second enzyme of the ether -lipid biosynthetic pathway which is responsible for the introduction of th e ether linkage between a fatty alcohol and a glycerol present in a subclas s of phospholipids, the plasmalogens and possibly in glycolipid membrane an chors. In this study the gene coding for alkyl dihydroxyacetone-phosphate s ynthase was isolated from Trypanosoma brucei. Southern blot analysis of tot al genomic DNA suggested the presence of a single copy gene. The analysis, together with sequencing of different cDNA clones showed that the two allel es of the gene differ in only one nucleotide. The gene encodes a protein of 612 amino acids with a calculated molecular mass of 68.891, not counting t he initiator methionine. It carries a type-1 peroxisomal targeting signal ( a C-terminal tripeptide - AHL) and a calculated overall positive charge of + 10. The gene was expressed in a bacterial system and the corresponding pr otein carrying a His-lag was purified. The recombinant alkyl dihydroxyaceto ne-phosphate synthase and the enzyme isolated directly from the glycosomes of bloodstream-form trypanosomes have comparable kinetics. The K-m for hexa decanol was 42 mu M, while approximately 100 mu M of palmitoyl dihydroxyace tone phosphate (DHAP) was necessary for optimal activity. Sodium chloride i nhibited both the His-tagged protein and the enzyme isolated from the glyco somes of bloodstream-form and insect stage T. brucei. (C) 1999 Elsevier Sci ence B.V. All rights reserved.