MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF MANNITOL-1-PHOSPHATASEFROM THE APICOMPLEXAN PARASITE EIMERIA-TENELLA

A metabolic pathway responsible for the biosynthesis and utilization o f mannitol is present in the seven species of Eimeria that infect chic kens, but is not in the avian host, Mannitol-l-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeuti c target, Amino acid sequence of tryptic peptides obtained from bioche mically purified Eimeria tenella M1Pase was used to synthesize degener ate oligonucleotide hybridization probes, Using these reagents, a part ial genomic clone and full-length cDNA clones have been isolated and c haracterized, The de deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine fami ly of phosphatases, This limited homology to other histidine phosphata ses does, however, include several conserved residues that have been s hown to be essential for their catalytic activity, Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E., tenella, M oreover, recombinant M1Pase is subject to active site directed, hydrox ylamine-reversible inhibition by the histidine-selective acylating rea gent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.