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. 1984 Jan;81(1):115–119. doi: 10.1073/pnas.81.1.115

Location of amino acid alterations in mutants of aspartate transcarbamoylase: Structural aspects of interallelic complementation.

H K Schachman, C D Pauza, M Navre, M J Karels, L Wu, Y R Yang
PMCID: PMC344621  PMID: 6364131

Abstract

Recent genetic studies of the pyrB locus of Escherichia coli resulted in the characterization of 29 mutant strains harboring defects in the structural gene that encodes the catalytic chains of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2). Three alleles, pyrB554, pyrB730, and pyrB748, have been cloned, and their nucleotide sequences have been determined along with that of the wild-type pyrBI operon in order to locate the sites of the alterations in the catalytic chains. Missense mutation pyrB554 leads to replacement of serine-52 by phenylalanine, and the inactive mutant enzyme has properties similar to those of wild-type aspartate transcarbamoylase. The amber mutation pyrB730 results in unstable truncated polypeptide chains 27 amino acids shorter than wild-type chains. Deletion mutation pyrB748 causes the removal of 181 amino acids. Combining these results with knowledge of the crystallographic structure of the wild-type enzyme provides a basis for tentative structural mechanisms for the observed complementation behavior of the mutant proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blackburn M. N., Schachman H. K. Allosteric regulation of aspartate transcarbamoylase. Effect of active site ligands on the reactivity of sulfhydryl groups of the regulatory subunits. Biochemistry. 1977 Nov 15;16(23):5084–5091. doi: 10.1021/bi00642a022. [DOI] [PubMed] [Google Scholar]
  2. CRICK F. H., ORGEL L. E. THE THEORY OF INTER-ALLELIC COMPLEMENTATION. J Mol Biol. 1964 Jan;8:161–165. doi: 10.1016/s0022-2836(64)80156-x. [DOI] [PubMed] [Google Scholar]
  3. Davies G. E., Vanaman T. C., Stark G. R. Aspartate transcarbamylase. Stereospecific restrictions on the binding site for L-aspartate. J Biol Chem. 1970 Mar 10;245(5):1175–1179. [PubMed] [Google Scholar]
  4. Gibbons I., Flatgaard J. E., Schachman H. K. Quaternary constraint in hybrid of aspartate transcarbamylase containing wild-type and mutant catalytic subunits. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4298–4302. doi: 10.1073/pnas.72.11.4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gigot D., Glansdorff N., Legrain C., Piérard A., Stalon V., Konigsberg W., Caplier I., Strosberg A. D., Hervé G. Comparison of the N-terminal sequences of aspartate and ornithine carbamoyltransferases of Escherichia coli. FEBS Lett. 1977 Sep 1;81(1):28–32. doi: 10.1016/0014-5793(77)80920-4. [DOI] [PubMed] [Google Scholar]
  6. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Honzatko R. B., Crawford J. L., Monaco H. L., Ladner J. E., Ewards B. F., Evans D. R., Warren S. G., Wiley D. C., Ladner R. C., Lipscomb W. N. Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli. J Mol Biol. 1982 Sep 15;160(2):219–263. doi: 10.1016/0022-2836(82)90175-9. [DOI] [PubMed] [Google Scholar]
  8. Honzatko R. B., Lipscomb W. N. Interactions of phosphate ligands with Escherichia coli aspartate carbamoyltransferase in the crystalline state. J Mol Biol. 1982 Sep 15;160(2):265–286. doi: 10.1016/0022-2836(82)90176-0. [DOI] [PubMed] [Google Scholar]
  9. Hoover T. A., Roof W. D., Foltermann K. F., O'Donovan G. A., Bencini D. A., Wild J. R. Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli. Proc Natl Acad Sci U S A. 1983 May;80(9):2462–2466. doi: 10.1073/pnas.80.9.2462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. JOVIN T., CHRAMBACH A., NAUGHTON M. A. AN APPARATUS FOR PREPARATIVE TEMPERATURE-REGULATED POLYACRYLAMIDE GEL ELECTROPHORESIS. Anal Biochem. 1964 Nov;9:351–369. doi: 10.1016/0003-2697(64)90192-7. [DOI] [PubMed] [Google Scholar]
  11. Jenness D. D., Schachman H. K. Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase. J Biol Chem. 1983 Mar 10;258(5):3266–3279. [PubMed] [Google Scholar]
  12. Jenness D. D., Schachman H. K. pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium. J Bacteriol. 1980 Jan;141(1):33–40. doi: 10.1128/jb.141.1.33-40.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Justesen J., Neuhard J. pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes. J Bacteriol. 1975 Sep;123(3):851–854. doi: 10.1128/jb.123.3.851-854.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Konigsberg W. H., Henderson L. Amino acid sequence of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli. Proc Natl Acad Sci U S A. 1983 May;80(9):2467–2471. doi: 10.1073/pnas.80.9.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Landfear S. M., Evans D. R., Lipscomb W. N. Elimination of cooperativity in aspartate transcarbamylase by nitration of a single tyrosine residue. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2654–2658. doi: 10.1073/pnas.75.6.2654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  17. Monaco H. L., Crawford J. L., Lipscomb W. N. Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5276–5280. doi: 10.1073/pnas.75.11.5276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Navre M., Schachman H. K. Synthesis of aspartate transcarbamoylase in Escherichia coli: transcriptional regulation of the pyrB-pyrI operon. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1207–1211. doi: 10.1073/pnas.80.5.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pauza C. D., Karels M. J., Navre M., Schachman H. K. Genes encoding Escherichia coli aspartate transcarbamoylase: the pyrB-pyrI operon. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4020–4024. doi: 10.1073/pnas.79.13.4020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith G. E., Summers M. D. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. doi: 10.1016/0003-2697(80)90019-6. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Staden R. Sequence data handling by computer. Nucleic Acids Res. 1977 Nov;4(11):4037–4051. doi: 10.1093/nar/4.11.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Syvanen J. M., Roth J. R. Structural genes for catalytic and regulatory subunits of aspartate transcarbamylase. J Mol Biol. 1973 May 25;76(3):363–378. doi: 10.1016/0022-2836(73)90510-x. [DOI] [PubMed] [Google Scholar]
  25. Wall K. A., Flatgaard J. E., Schachman H. K., Gibbons I. Purification and characterization of a mutant aspartate transcarbamoylase lacking enzyme activity. J Biol Chem. 1979 Dec 10;254(23):11910–11916. [PubMed] [Google Scholar]
  26. Wall K. A., Schachman H. K. Primary structure and properties of an inactive mutant aspartate transcarbamoylase. J Biol Chem. 1979 Dec 10;254(23):11917–11926. [PubMed] [Google Scholar]
  27. Weber K. New structural model of E. coli aspartate transcarbamylase and the amino-acid sequence of the regulatory polypeptide chain. Nature. 1968 Jun 22;218(5147):1116–1119. doi: 10.1038/2181116a0. [DOI] [PubMed] [Google Scholar]
  28. Yang Y. R., Syvanen J. M., Nagel G. M., Schachman H. K. Aspartate transcarbamoylase molecules lacking one regulatory subunit. Proc Natl Acad Sci U S A. 1974 Mar;71(3):918–922. doi: 10.1073/pnas.71.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zabin I., Villarejo M. R. Protein complementation. Annu Rev Biochem. 1975;44:295–313. doi: 10.1146/annurev.bi.44.070175.001455. [DOI] [PubMed] [Google Scholar]
  30. Zabin I. beta-Galactosidase alpha-complementation. A model of protein-protein interaction. Mol Cell Biochem. 1982 Nov 26;49(2):87–96. doi: 10.1007/BF00242487. [DOI] [PubMed] [Google Scholar]

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