title: Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli.
abstract: The phosphoenolpyruvate mutase gene from Tetrahymena pyriformis has been cloned and overexpressed in Escherichia coli. To our knowledge, this is the first Tetrahymena gene to be expressed in E. coli, a task made more complicated by the idiosyncratic codon usage by Tetrahymena. The N-terminal amino acid sequence of phosphoenolpyruvate mutase purified from T. pyriformis has been used to generate a precise oligonucleotide probe for the gene, using in vitro amplification from total genomic DNA by the polymerase chain reaction. Use of this precise probe and oligo(T) as primers for in vitro amplification from a T. pyriformis cDNA library has allowed the cloning of the mutase gene. A similar amplification strategy from genomic DNA yielded the genomic sequence, which contains three introns. The sequence of the DNA that encodes 10 amino acids upstream of the N-terminal sequence of the isolated protein was found by oligonucleotide hybridization to a subgenomic library. These 10 N-terminal amino acids are cleanly removed in Tetrahymena in vivo. The full mutase gene sequence codes for a protein of 300 amino acids, and it includes two amber (TAG) codons in the open reading frame. In Tetrahymena, TAG codes for glutamine. When the two amber codons are each changed to a glutamine codon (CAG) that is recognized by E. coli and the gene is placed behind a promoter driven by the T7 RNA polymerase, expression in E. coli is observed. The mutase gene also contains a large number of arginine AGA codons, a codon that is very rarely used by E. coli. Cotransformation with a plasmid carrying the dnaY gene [which encodes tRNA(Arg)(AGA)] results in more than 4-fold higher expression. The mutase then comprises about 25% of the total soluble cell protein in E. coli transformants. The mutase gene bears significant similarity to one other gene in the available data bases, that of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus, an enzyme that catalyzes a closely related transformation. Due to the large evolutionary distance between Tetrahymena and Streptomyces, this similarity can be interpreted as the first persuasive evidence that the biosynthesis of phosphonates is an ancient metabolic process.
authors.affiliation: Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
authors.forename: H M
authors.initials: HM
authors.lastname: Seidel
authors.forename: D L
authors.initials: DL
authors.lastname: Pompliano
authors.forename: J R
authors.initials: JR
authors.lastname: Knowles
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005973:Glutamine / Q000235:genetics; D007438:Introns; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D063065:Organophosphonates / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases); D016133:Polymerase Chain Reaction; D013769:Tetrahymena pyriformis / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D003062:Codon; D015345:Oligonucleotide Probes; D063065:Organophosphonates; D005973:Glutamine; D007535:Isomerases; D017875:Phosphotransferases (Phosphomutases); C057167:phosphoenolpyruvate mutase