Molecular cloning and functional expression of mannitol-1-phosphatase from the apicomplexan parasite Eimeria tenella

• Published in: The Journal of biological chemistry Vol. 273; no. 7; pp. 4237 - 4244
• Main Authors: Liberator, P, Anderson, J, Feiglin, M, Sardana, M, Griffin, P, Schmatz, D, Myers, R.W
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 13.02.1998
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN; Amino Acid Sequence; AMINO ACID SEQUENCES; analysis; Animals; Base Sequence; Binding Sites; CHEMICAL COMPOSITION; chemistry; CLONACION; CLONAGE; CLONING; Cloning, Molecular; COMPLEMENTARY DNA; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DIETHYL PYROCARBONATE; Diethyl Pyrocarbonate - pharmacology; DNA; EIMERIA TENELLA; Eimeria tenella - enzymology; enzyme activity; ENZYME INHIBITORS; ENZYMIC ACTIVITY; enzymology; ESTERASAS; ESTERASE; ESTERASES; GENBANK/AF032462; Gene Expression; Gene Expression - genetics; genetics; HISTIDINA; HISTIDINE; INHIBICION; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; INHIBITION; Kinetics; metabolism; MOLECULAR SEQUENCE DATA; NUCLEOTIDE SEQUENCE; nucleotide sequences; Peptide Fragments; Peptide Fragments - analysis; pharmacology; PHOSPHORIC MONOESTER HYDROLASES; Phosphoric Monoester Hydrolases - chemistry; Recombinant Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SECUENCIA NUCLEOTIDICA; Sequence Analysis, DNA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; Trypsin; Trypsin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.7.4237

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown 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 . Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.