From Biological Diversity to Structure-Function Analysis: Protein Engineering in Aspartate Transcarbamoylase

  • James R. Wild
  • Janet K. Grimsley
  • Karen M. Kedzie
  • Melinda E. Wales
Part of the Industry-University Cooperative Chemistry Program Symposia book series (IUCC)

Summary

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is a common enzyme which catalyzes the first unique step in pyrimidine biosynthesis in divergent biological systems; however, it possesses tremendous architectural variety from one organism to another. For example, the E. coli ATCase holoenzyme is comprised of two catalytic trimers and three regulatory dimers, while the mammalian enzyme is part of a multifunctional protein aggregate encoding the preceding and subsequent enzymes in pyrimidine biosynthesis. Despite extreme differences in quaternary architecture and enzymatic organization, protein engineering studies have demonstrated the existence of highly conserved units of protein structure that impart specific functional characteristics.
  1. 1

    The largest of these units are discrete polypeptides or superdomains within multifunctional proteins which have been shown to be uniquely involved in specific catalytic steps within the CAD or CA complexes of eukaryotic pyrimidine biosynthesis.

     
  2. 2

    The catalytic polypeptides of various ATCases are organized into two discrete and separable binding domains for its substrates, carbamoyl phosphate and aspartate.

     
  3. 3

    The regulatory polypeptides of the enteric bacterial enzymes also contain two discrete tertiary domains, the Allosteric Binding Domain and Cys 4 coordinated Zinc Domain involved in the protein:protein interface between the regulatory and catalytic polypeptides of the holoenzyme.

     
  4. 4

    There are sub-domain structural units within the various polypeptides which have a coordinated impact on specific catalytic and regulatory functions in the enzyme.

     
  5. 5

    Finally, it has been possible to ascribe some individual function to specific amino acids relative to ligand binding, zinc coordination, protein:protein interactions, and the structural reorganizations in the T-R transition of the enteric holoenzymes.

     

Comparisons of the catalytic sequences of various ATCases have revealed substantial conservation of primary and predicted secondary structures. Based upon the sequence alignment and the E. coli ATCase crystal structure, the hamster ATCase superdomain tertiary structure has been modeled with interactive computer graphics. The predicted conservation of structure/function relationships were verified experimentally through the construction of active catalytic bacterial/hamster chimeric enzymes. It has also been possible to verify the apparent structural homologies of ATCase and ornithine transcarbamoylase (OTCase, 2.1.3.3), the parallel enzyme from arginine biosynthesis, by exchange of the amino acid binding domains of the two enzymes. Extending these observations and protein engineering philosophies into the formation of hybrid and chimeric enzymes has resulted in the production of ATCases with altered catalytic and regulatory characteristics.

Keywords

Regulatory Subunit Pyrimidine Biosynthesis Chimeric Enzyme Zinc Binding Domain Hybrid Enzyme 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    J.R. Wild and M.E. Wales. Molecular Evolution and Genetic Engineering of Protein Domains Involving Aspartate Transcarbamoylase. Ann. Rev. Microbiol. 44:93-118.Google Scholar
  2. 2.
    D.R. Evans. CAD, a chimeric protein that initiates de novo pyrimidine biosynthesis in higher eukaryotes. In Multidomain proteins-structure and evolution. ed. D.G. Hardie, J.R. Coggins, pp. 283–331. New York: Elsevier. (1986).Google Scholar
  3. 3.
    M.E. Jones. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Ann. Rev. Biochem. 49:253–79 (1980).PubMedCrossRefGoogle Scholar
  4. 4.
    J.N. Freund and B.P. Jarry. 1987. The rudimentary gene of Drosophila melanogaster encodes four enzymatic functions. J. Mol. Biol. 193:1–13 (1987).PubMedCrossRefGoogle Scholar
  5. 5.
    J.N. Davidson, P.C. Rumsby and J. Tamaren. Organization of a multifunctional protein in pyrimidine biosynthesis. J. Biol. Chem. 256:5220-5225.Google Scholar
  6. 6.
    G.A. O’Donovan and J. Neuhard. Pyrimidine metabolism in microorganisms. Bacteriol. Rev. 34:278–343 (1970).PubMedGoogle Scholar
  7. 7.
    A.J. Makoff and A. Radford. Genetics and biochemistry of carbamoyl phosphate biosynthesis and its utilization in the pyrimidine bisoynthetic pathway. Microbiol. Rev. 42:307-28.Google Scholar
  8. 8.
    J-L. Souciet, M. Nagy, M. LeGouar, F. LaCroute, S. Potier. Organization of the yeast URA2 gene: identification of a defective dihydroorotase like domain in the multifunctional carbamoylphosphate synthetase aspartate transcarbamylase complex. Gene (Amst.) 79:59–70 (1989).CrossRefGoogle Scholar
  9. 9.
    M. Faure, J.H. Camonis and M. Jacquet. Molecular characterization of a Dictyostelium discoideum gene encoding a multifunctional enzyme of the pyrimidine pathway. Eur. J. Biochem. 179:345–58 (1989).PubMedCrossRefGoogle Scholar
  10. 10.
    K.L. Krause, K.W. Volz and W.N. Lipscomb, W.N. 2.5A structure of aspartate carbamoyl transferase complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate. J. Mol. Biol. 193:527–53.75 (1987).PubMedCrossRefGoogle Scholar
  11. 11.
    J.E. Gouaux and W.N. Lipscomb. The three dimensional structure of carbamyl phosphate and succinate bound to aspartate carbamoyltransferase. Proc. Natl. Acad. Sci. USA 85:4205–4208 (1988).PubMedCrossRefGoogle Scholar
  12. 12.
    E.R. Kantrowitz and W.N. Lipscomb. Escherichia coli aspartate transcarbamoylase: the relation between structure and function. Science 241:669–74 (1988).PubMedCrossRefGoogle Scholar
  13. 13.
    H.M. Ke. R.B. Honzatko and W.N. Lipscomb. Structure of unligated aspartate carbamoyltransferase of Escherichia coli at 2.6 A resolution. Proc. Natl. Acad. Sci. USA 81:4037–40 (1984).PubMedCrossRefGoogle Scholar
  14. 14.
    K.H. Kim, Z. Pan, R.B. Honzatko, H.M. Ke and W.N. Lipscomb. Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli. J. Mol. Biol. 196:853–75 (1987).PubMedCrossRefGoogle Scholar
  15. 15.
    M.R. Bethell and M.E. Jones. Molecular size and feedback-regulation characteristics of bacterial aspartate transcarbamoylases. Arch. Biochem. Biophvs. 134:352–65 (1967).CrossRefGoogle Scholar
  16. 16.
    J.R. Wild, W.L. Belser, W.L. O’Donovan and GA. O’Donovan. Unique aspects of the regulation of the aspartate transcarbamoylase of Serratia marcescens. J. Bacteriol. 28:766–75 (1976).Google Scholar
  17. 17.
    J.R. Wild, K.F. Foltermann and GA. O’Donovan. Regulatory divergence of aspartate transcarbamoylase within the Enterobacteriaceae. Arch. Biochem. Biophvs. 201:506–17 (1980).CrossRefGoogle Scholar
  18. 18.
    K.F. Foltermann, J.R. Wild, D.L. Zink and GA. O’Donovan, GA. Regulatory variance of aspartate transcarbamoylase among strains of Yersinia entercolitica and Yersinia entercolitica-like organisms. Curr. Microbiol. 6:43–47.71 (1981).CrossRefGoogle Scholar
  19. 19.
    D-A. Beck, K.M. Kedzie and J.R. Wild. Comparison of the aspartate transcarbamoylases from Serratia marcescens and Escherichia coli. J. Biol. Chem. 264:16629–37 (1989).PubMedGoogle Scholar
  20. 20.
    J.S. Brabson and R.L. Switzer, Purification and properties of Bacillus subtilis aspartate transcarbamoylase. J. Biol. Chem. 250:8664–69 (1975).PubMedGoogle Scholar
  21. 21.
    CG. Lerner and R.L. Switzer. Cloning and Structure of the Bacillus subtilis aspartate transcarbamoylase gene (pyrB). J. Biol. Chem. 261:11156–65 (1986).PubMedGoogle Scholar
  22. 22.
    L.B. Adair and M.E. Jones. Purification and characteristics of aspartate transcarbamoylase from Pseudomonas fluroescens. J. Biol. Chem. 247:2308–2315 (1972).PubMedGoogle Scholar
  23. 23.
    S.T. Berth and D.R. Evans. Characterization of the aspartate transcarbamoylase of Pseudomonas fluorescens. FASEB J. 4A1836.Google Scholar
  24. 24.
    F. Van Vliet, R. Cunin, A. Jacobs, J. Piette, and D. Gigot. Evolutionary divergence of genes for ornithine and aspartate carbamoyltransferases-complete sequence and mode of regulation of the Escherichia coli argF with pyrB. Nucl. Acids Res. 12:6277–89 (1984).PubMedCrossRefGoogle Scholar
  25. 25.
    D.A. Bencini, J.E. Houghton, T.A. Hoover, K.F. Foltermann and J.R. Wild. The DNA sequence of argl from Escherichia coli K12. Nucleic Acids Res. 11:8509–18 (1983).PubMedCrossRefGoogle Scholar
  26. 26.
    J.E. Houghton, D.A. Bencini, G.A. O’Donovan and J.R. Wild. Protein differentiation: a comparison of aspartate transcarbamoylase and ornithine transcarbamoylase from Escherichia coli K-12. Proc. Natl. Acad. Sci. USA 81:4864–68 (1984).PubMedCrossRefGoogle Scholar
  27. 27.
    Y. Itoh, L. Soldati, V. Stalon, P. Falmagne, and Y. Terawaki. Anabolic ornithine carbamoyltransferase of Pseudomonas aeurginosa: nucleotide sequence and transcriptional control of the argF gene. J. Bacteriol. 170:2725–34 (1988).PubMedGoogle Scholar
  28. 28.
    J.A. Maley and J.N. Davidson, The aspartate transcarbamoylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli. Mol. Gen. Genet. 213:278–84 (1988).PubMedCrossRefGoogle Scholar
  29. 29.
    J.G. Major. Structural modelling of the hamster CAD aspartate transcarbamoylase and comparisons to the Escherichia coli ATCase trimer. PhD Dissertation, Texas A&M Univ. College Station. 122 pp (1989).Google Scholar
  30. 30.
    J.G. Major, M.E. Wales, J.E. Houghton, J.A. Maley, J.N. Davidson and J.R. Wild. Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase. J. Mol. Evol. 28:442–450 (1989).PubMedCrossRefGoogle Scholar
  31. 31.
    J.G. Major, R. Radhakrishnan, J. Villano, E.F. Meyer and J.R. Wild. Structural modeling of the hamster CAD aspartate transcarbamoylase superdomain and comparisons to the Escherichia coli ATCase trimer, (submitted for publication).Google Scholar
  32. 32.
    J.E. Houghton. Structural and functional comparisons between OTCase and ATCase leading to the formation and characterization of an active domain fusion between argI (OTCase) and pyrB (ATCase). PhD Dissertation, Texas A&M University, College Station. 140 pp (1986).Google Scholar
  33. 33.
    J.E. Houghton, G.A. O’Donovan and J.R. Wild. Reconstruction of an enzyme by domain substitution effectively switches substrate specificity. Nature 338:172–74 (1989).PubMedCrossRefGoogle Scholar
  34. 34.
    K.F. Foltermann, M.S. Shanley, and J.R. Wild. Assembly of the aspartate transcarbamoylase holoenzyme from transcriptionally independent catalytic and regulatory cistrons. J. Bacteriol. 157:891–98 (1984).PubMedGoogle Scholar
  35. 35.
    M.S. Shanley, K.F. Foltermann, G.A. O’Donovan and J.R. Wild. Properties of hybrid aspartate transcarbamoylase with native subunits from divergent bacteria. J. Biol. Chem. 259:12672–77 (1984).PubMedGoogle Scholar
  36. 36.
    K.F. Foltermann, D-A. Beck and J.R. Wild. In vivo formation of hybrid aspartate transcarbamoylases from native subunits of divergent members of the family Enterobacteriaceae. J. Bacteriol. 167:285–90 (1986).PubMedGoogle Scholar
  37. 37.
    K.M. Kedzie. Characterization of the pyrB gene of Serratia marcescens and hybrid gene formation with the pyrB gene of Escherichia coli, leading to the production of chimeric ATCase. Ph.D Dissertation, Texas A&M Univ., College Station. 158 pp (1987).Google Scholar
  38. 38.
    K.M. Kedzie, GA. O’Donovan and J.R. Wild. In preparation.Google Scholar
  39. 39.
    R. Cunin, A. Jacobs, D. Charlier, M. Crabeel, and G. Hérve. Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli I. Primary structure of a pyrI gene encoding a modified regulatory subunit. J. Mol. Biol. 186:707–13 (1985).PubMedCrossRefGoogle Scholar
  40. 40.
    M.E. Wales, J. Moehlman and J.R. Wild. Characterization of a exchange fusion aspartate transcarbamoylase involving the 200s loop of the catalytic subunit which effects the T-R transition. (submitted for publication).Google Scholar
  41. 41.
    J.K. Grimsley. Role of the 200s loop in the heterotropic and homotropic response of Escherichia coli aspartate transcarbamoylase. PhD Dissertation. Texas A&M University, College Station, Texas. 99pp (1989).Google Scholar
  42. 42.
    T.S. Corder and J.R. Wild. Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site. J. Biol. Chem. 264:7425–7430 (1989).PubMedGoogle Scholar
  43. 43.
    M.E. Wales. TA. Hoover and J.R. Wild. Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme. J. Biol. Chem. 263:6109–14 (1988).PubMedGoogle Scholar
  44. 44.
    M.E. Wales and J.R. Wild. Role of the 240s loop interactions in stabilizing catalytic site domain closure in defining kinetic parameters of aspartate transcarbamoylase. (submitted for publication).Google Scholar
  45. 45.
    H. Nyunoya, K.E. Broglie and C.J. Lusty, The gene coding for carbamoyl-phosphate synthetase I was formed by fusion of an ancestral glutaminase gene and a synthetase gene. J. Biol. Chem. 259:9790–98 (1985).Google Scholar
  46. 46.
    G.A. O’Donovan, H. Holoubek and J.C. Gerhart. Regulatory properties of intergeneric hybrids of aspartate transcarbamoylase. Nature New Biol. 238:264–266 (1972).PubMedGoogle Scholar
  47. 47.
    J.K. Grimsley, M.E. Wales and J.R. Wild. Role of the 200s loop of the catalytic polypeptide in transmitting the homotropic and heterotropic responses of aspartate transcarbamoylase. (submitted for publication).Google Scholar
  48. 48.
    J.C. Gerhart and A.B. Pardee. The enzymology of control by feedback inhibition. J. Biol. Chem. 237:891–96 (1962).PubMedGoogle Scholar
  49. 49.
    J. Monod, J. Wyman and J-P. Changeux. On the nature of allosteric transition: A plausible model. J. Mol. Biol. 12:88–118 (1965).PubMedCrossRefGoogle Scholar
  50. 50.
    H.K. Schachman. Can a simple model account for the allosteric transition of aspartate transcarbamoylase? J. Biol. Chem. 263:18583–18586 (1988).PubMedGoogle Scholar
  51. 51.
    J.R. Wild, S J. Loughrey and T.C. Corder. In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase. Proc. Natl. Acad. Sci. USA. 86:52–56 (1989).CrossRefGoogle Scholar
  52. 52.
    J.R. Wild, J.L. Johnson and S J. Loughrey. ATP-liganded form of aspartate transcarbamoylase, the logical regulatory target for allosteric control in divergent bacterial systems. J. Bacteriol. 28:766–75 (1988).Google Scholar
  53. 53.
    K.F. Foltermann, W. Lagaly and J.R. Wild. The aspartate transcarbamoylase from Proteus vulgaris: feedback inhibition requires CTP/UTP synergism. (submitted for publication).Google Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • James R. Wild
    • 1
  • Janet K. Grimsley
    • 1
  • Karen M. Kedzie
    • 1
  • Melinda E. Wales
    • 1
  1. 1.Department of Biochemistry and BiophysicsTexas A&M University SystemCollege StationUSA

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