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. 1996 Apr;5(4):719–728. doi: 10.1002/pro.5560050417

Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: implications for domain switching.

L B Murata 1, H K Schachman 1
PMCID: PMC2143375  PMID: 8845762

Abstract

Each catalytic (c) polypeptide chain of Escherichia coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helices. Helix 12, near the C-terminus, extends from the second domain back through the first domain, bringing the two termini close together. This helix is of critical importance for the assembly of a stable enzyme. The trimeric E. coli enzyme ornithine transcarbamoylase (OTCase) is proposed to be similar in tertiary and quaternary structure to the ATCase trimer and has a predicted alpha-helical segment near its C-terminus. In our companion paper, we have shown that this putative helix is essential for OTCase folding and assembly (Murata L, Schachman HK, 1996, Protein Sci 5:709-718). Here, the similarity between OTCase and the ATCase trimer, which are 32% identical in sequence, was tested further by the construction of several chimeras in which various structural elements were switched between the enzymes by genetic techniques. These elements included the two globular domains and regions containing the C-terminal helices. In contrast to results reported previously (Houghton J, O'Donovan G, Wild J, 1989, Nature 338:172-174), none of the chimeric proteins exhibited in vivo activity and all were insoluble when overexpressed. Attempts to make hybrid trimers composed of c chains from ATCase and OTCase were also unsuccessful. These results underscore the complexities of specific intrachain and interchain side-chain interactions required to maintain tertiary and quaternary structures in these enzymes.

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Selected References

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  1. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Houghton J. E., O'Donovan G. A., Wild J. R. Reconstruction of an enzyme by domain substitution effectively switches substrate specificity. Nature. 1989 Mar 9;338(6211):172–174. doi: 10.1038/338172a0. [DOI] [PubMed] [Google Scholar]
  5. Ke H. M., Lipscomb W. N., Cho Y. J., Honzatko R. B. Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms. J Mol Biol. 1988 Dec 5;204(3):725–747. doi: 10.1016/0022-2836(88)90365-8. [DOI] [PubMed] [Google Scholar]
  6. Kim K. H., Pan Z. X., Honzatko R. B., Ke H. M., Lipscomb W. N. Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli. J Mol Biol. 1987 Aug 20;196(4):853–875. doi: 10.1016/0022-2836(87)90410-4. [DOI] [PubMed] [Google Scholar]
  7. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  8. Major J. G., Jr, Wales M. E., Houghton J. E., Maley J. A., Davidson J. N., Wild J. R. Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase. J Mol Evol. 1989 May;28(5):442–450. doi: 10.1007/BF02603079. [DOI] [PubMed] [Google Scholar]
  9. Mas M. T., Chen C. Y., Hitzeman R. A., Riggs A. D. Active human-yeast chimeric phosphoglycerate kinases engineered by domain interchange. Science. 1986 Aug 15;233(4765):788–790. doi: 10.1126/science.3526552. [DOI] [PubMed] [Google Scholar]
  10. Mendoza J. A., Rogers E., Lorimer G. H., Horowitz P. M. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J Biol Chem. 1991 Jul 15;266(20):13044–13049. [PubMed] [Google Scholar]
  11. Moe G. R., Bollag G. E., Koshland D. E., Jr Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5683–5687. doi: 10.1073/pnas.86.15.5683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  13. Murata L. B., Schachman H. K. Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase. Protein Sci. 1996 Apr;5(4):709–718. doi: 10.1002/pro.5560050416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Newton C. J., Stevens R. C., Kantrowitz E. R. Importance of a conserved residue, aspartate-162, for the function of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1992 Mar 24;31(11):3026–3032. doi: 10.1021/bi00126a026. [DOI] [PubMed] [Google Scholar]
  15. Peterson C. B., Schachman H. K. Role of a carboxyl-terminal helix in the assembly, interchain interactions, and stability of aspartate transcarbamoylase. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):458–462. doi: 10.1073/pnas.88.2.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Piette J., Cunin R., Van Vliet F., Charlier D., Crabeel M., Ota Y., Glansdorff N. Homologous control sites and DNA transcription starts in the related argF and argI genes of Escherichia coli K12. EMBO J. 1982;1(7):853–857. doi: 10.1002/j.1460-2075.1982.tb01259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Richardson J. S. The anatomy and taxonomy of protein structure. Adv Protein Chem. 1981;34:167–339. doi: 10.1016/s0065-3233(08)60520-3. [DOI] [PubMed] [Google Scholar]
  18. Roof W. D., Foltermann K. F., Wild J. R. The organization and regulation of the pyrBI operon in E. coli includes a rho-independent attenuator sequence. Mol Gen Genet. 1982;187(3):391–400. doi: 10.1007/BF00332617. [DOI] [PubMed] [Google Scholar]
  19. Schachman H. K., Pauza C. D., Navre M., Karels M. J., Wu L., Yang Y. R. Location of amino acid alterations in mutants of aspartate transcarbamoylase: Structural aspects of interallelic complementation. Proc Natl Acad Sci U S A. 1984 Jan;81(1):115–119. doi: 10.1073/pnas.81.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schneider W. P., Nichols B. P., Yanofsky C. Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2169–2173. doi: 10.1073/pnas.78.4.2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stevens R. C., Chook Y. M., Cho C. Y., Lipscomb W. N., Kantrowitz E. R. Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis. Protein Eng. 1991 Apr;4(4):391–408. doi: 10.1093/protein/4.4.391. [DOI] [PubMed] [Google Scholar]
  22. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thornton J. M., Sibanda B. L. Amino and carboxy-terminal regions in globular proteins. J Mol Biol. 1983 Jun 25;167(2):443–460. doi: 10.1016/s0022-2836(83)80344-1. [DOI] [PubMed] [Google Scholar]
  24. Van Vliet F., Cunin R., Jacobs A., Piette J., Gigot D., Lauwereys M., Piérard A., Glansdorff N. Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB. Nucleic Acids Res. 1984 Aug 10;12(15):6277–6289. doi: 10.1093/nar/12.15.6277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wetlaufer D. B. Nucleation, rapid folding, and globular intrachain regions in proteins. Proc Natl Acad Sci U S A. 1973 Mar;70(3):697–701. doi: 10.1073/pnas.70.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wild J. R., Wales M. E. Molecular evolution and genetic engineering of protein domains involving aspartate transcarbamoylase. Annu Rev Microbiol. 1990;44:193–218. doi: 10.1146/annurev.mi.44.100190.001205. [DOI] [PubMed] [Google Scholar]
  27. Wong H., Davis R. C., Nikazy J., Seebart K. E., Schotz M. C. Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11290–11294. doi: 10.1073/pnas.88.24.11290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yang Y. R., Schachman H. K. Hybridization as a technique for studying interchain interactions in the catalytic trimers of aspartate transcarbamoylase. Anal Biochem. 1987 May 15;163(1):188–195. doi: 10.1016/0003-2697(87)90111-4. [DOI] [PubMed] [Google Scholar]

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