1887

Abstract

A cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by . The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the gene cloned from was deleted in with exonuclease III and fused in-frame with a 5' portion of to yield a new gene, denoted *, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the α-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the of the TyrA* dehydrogenase for NADremained unaltered, the for prephenate was fourfold greater and the was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by * was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the value for L-tyrosine was decreased from 66 μM to 14 μM. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein. These include alterations in apparent substrate specificity, isoelectric point, stability, catalytic properties and regulatory properties.

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1992-07-01
2021-04-21
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References

  1. Ahmad S., Jensen R. A. 1986; The evolutionary history of two bifunctional proteins that emerged in the purple bacteria. Trends in Biochemical Sciences 11 108 112
    [Google Scholar]
  2. Ahmad S., Jensen R. A. 1987; The prephenate dehydrogenase component of the bifunctional T-protein in enteric bacteria can utilize l-arogenate. FEBS Letters 216 133 139
    [Google Scholar]
  3. Ahmad S., Jensen R. A. 1988; Phylogenetic distribution of components of the overflow pathway to l-phenylalanine within the enteric lineage of bacteria. Current Microbiology 16 295 302
    [Google Scholar]
  4. Ahmad S., Weisburg W. G., Jensen R. A. 1990; Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria. Journal of Bacteriology 172 1051 1061
    [Google Scholar]
  5. Baldwin G. S., Davidson B. E. 1981; A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K-12 defective in the pheA gene. Archives of Biochemistry and Biophysics 211 66 75
    [Google Scholar]
  6. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248 254
    [Google Scholar]
  7. Byng G. S., Whitaker R. J., Gherna R. L., Jensen R. A. 1980; Variable enzymological patterning in tyrosine biosynthesis as a means of determining natural relatedness among the Pseudomonadaceae. Journal of Bacteriology 144 247 257
    [Google Scholar]
  8. Cotton R. G. H., Gibson F. 1965; The biosynthesis of phenylalanine and tyrosine: enzymes converting chorismic acid into prephenic acid and their relationships to prephenate dehydratase and prephenate dehydrogenase. Biochimica et Biophysica Acta 100 76 88
    [Google Scholar]
  9. Dayan J., Sprinson D. B. 1970; Preparation of prephenic acid. Methods in Enzymology 17A 559 561
    [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12 387 395
    [Google Scholar]
  11. Gibson F. 1964; Chorismic acid. Purification and some chemical and physical studies. Biochemical Journal 90 256 261
    [Google Scholar]
  12. Henikoff S. 1984; Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28 351 359
    [Google Scholar]
  13. Heyde E. 1979; Chorismate mutase–prephenate dehydrogenase from Aerobacter aerogenes: evidence that the two reactions occur at one active site. Biochemistry 18 2766 2775
    [Google Scholar]
  14. Heyde E., Morrison J. F. 1978; Kinetic studies on the reactions catalyzed by chorismate mutase–prephenate dehydrogenase from Aerobacter aerogenes. Biochemistry 17 1573 1580
    [Google Scholar]
  15. Hudson G. S., Davidson B. E. 1984; Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K-12. Journal of Molecular Biology 180 1023 1051
    [Google Scholar]
  16. Humphreys G. O., Willshaw G. A., Anderson E. S. 1975; A simple method for the preparation of large quantities of pure plasmid DNA. Biochimica et Biophysica Acta 383 457 463
    [Google Scholar]
  17. Koch G. L. E., Shaw D. C., Gibson F. 1972; Studies on the relationship between the active sites of chorismate mutase–prephenate dehydrogenase from Escherichia coli and Aerobacter aerogenes. Biochimica et Biophysica Acta 258 719 730
    [Google Scholar]
  18. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227 680 685
    [Google Scholar]
  19. Maruya A., O’Connor M. J., Backman K. 1987; Genetic separability of the chorismate mutase and prephenate dehydrogenase components of the Escherichia coli tyrA gene product. Journal of Bacteriology 169 4852 4853
    [Google Scholar]
  20. Prober J. M., Trainor G. L., Dam R. J., Hobbs F. W., Robertson C. W., Zagursky R. J., Cocuzza A. J., Jensen M. A., Baumeister K. 1987; A system for rapid DNA sequencing with fluorescent chain-terminating dideoxy nucleotides. Science 238 336 341
    [Google Scholar]
  21. Rood J. I., Perrot B., Heyde E., Morrison J. F. 1982; Characterization of monofunctional chorismate mutase/prephenate dehydrogenase enzymes obtained via mutagenesis of recombinant plasmids in vitro. European Journal of Biochemistry 124 513 519
    [Google Scholar]
  22. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Stewart J., Wilson D. B., Ganem B. 1990; A genetically engineered monofunctional chorsmate mutase. Journal of the American Chemical Society 112 4582 4584
    [Google Scholar]
  24. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Genetics 33 103 119
    [Google Scholar]
  25. Zamir L. O., Jensen R. A., Arison B. D., Douglas A. W., Albers S. G., Bowen J. R. 1980; Structure of arogenate (pretyrosine), an amino acid intermediate of aromatic biosynthesis. Journal of the American Chemical Society 102 4499 4504
    [Google Scholar]
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