Abstract
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.
Highlights
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
The sequence of several tryptic peptides from this purified enzyme has allowed us to isolate and characterize genomic and cDNA clones that code for E. tenella M1Pase
Cloning of the cDNA Encoding E. tenella M1Pase—Three degenerate antisense oligonucleotides were synthesized based upon sequence contained within peptides 21, 36, and 38
Summary
Materials—Oligonucleotides were synthesized using an Applied Biosystems model 384 DNA synthesizer and purified according to the manufacturer’s recommendations. Genomic DNA in this gel slice was extracted, ligated into ApaI-digested pBluescript KS (Stratagene), and used to transform electrocompetent DH10B bacteria This subgenomic plasmid library was screened by colony lift hybridization using end-labeled oligonucleotide 36-a. PCR reaction products were gel-purified, sequentially digested with NcoI and HindIII, directionally cloned as 5Ј-NcoI/3Ј-HindIII fragments into the bacterial expression vector pQE60 (Qiagen, Inc.), and used to transform electrocompetent bacteria. Fractions 33– 40 (nominal imidazole concentration of 65–110 mM) containing the major peak of purified M1Pase, as determined both by enzyme activity and Western blot immunoreactivity, were pooled, yielding 15.1 mg of protein from 0.56 g wet weight bacterial pellet. Bacterial cells expressing the nonfusion M1Pase construct (1.17 g wet weight) were collected as described above and were sonicated (6 ϫ 60 s) in 4 ml of buffer B (10 mM Hepes sodium, pH 7.4). Ions were detected throughout the entire LC gradient over a m/z (mass to charge ratio) range of 500 –2000
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
