Skip to main content
SpringerLink
Log in

Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

  • Published:
Journal of Molecular Evolution Aims and scope Submit manuscript

Summary

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB − cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adair LB, Jones ME (1972) Purification and characteristics of aspartate transcarbamylase fromPseudomonas fluorescens. J Biol Chem 247:2308–2315

    PubMed  CAS  Google Scholar 

  • Bethell MA, Smith KE, White JS, Jones ME (1968) Carbamyl phosphate: an allosteric substrate for aspartate transcarbamylase ofEscherichia coli. Proc Natl Acad Sci USA 60:1442–1449

    Article  PubMed  CAS  Google Scholar 

  • Blake C (1983) Exons and the evolution of proteins. Trends Biochem Sci 8:11–13

    Article  CAS  Google Scholar 

  • Brabson JS, Maurizi MR, Switzer RL (1985) Aspartate transcarbamylase fromBacillus subtilis. Methods Enzymol 113:627–635

    Article  PubMed  CAS  Google Scholar 

  • Carrey EA, Hardie DG (1988) Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. Eur J Biochem 171:583–588

    Article  PubMed  CAS  Google Scholar 

  • Casadaban MJ, Cohen SN (1979) Lactose genes fused to exogenous promoter in one step using Mu-lac bacteriophage.In vivo probe for transcription control sequences. Proc Natl Acad Sci USA 76:4530–4540

    Article  PubMed  CAS  Google Scholar 

  • Chang T, Jones ME (1974) Aspartate transcarbamylase fromStreptococcus faecalis. Purification, properties and nature of an allosteric activator site. Biochemistry 13:629–637

    Article  PubMed  CAS  Google Scholar 

  • Chen EJ, Seeburg PH (1985) Supercoil sequencing—a fast and simple method for sequencing plasmid DNA. DNA 4:165–170

    PubMed  CAS  Google Scholar 

  • Chou PY, Fasman GD (1974) Conformational parameters for amino acids in helical β-sheet, and random coil regions calculated from proteins. Biochemistry 13:211–245

    Article  PubMed  CAS  Google Scholar 

  • Chung CH, Goldberg AL (1981) The product of thelon (capR) gene inEscherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci USA 78:4931–4935

    Article  PubMed  CAS  Google Scholar 

  • Coleman PF, Suttle DP, Stark GR (1977) Purification from hamster cells of the multifunctional protein that initiatesde novo synthesis of pyrimidine nucleotides. J Biol Chem 252:6379–6385

    PubMed  CAS  Google Scholar 

  • Davidson JN, Niswander LA (1983) Partial cDNA sequence to a hamster gene corrects defect inEscherichia coli pyr B mutant. Proc Natl Acad Sci USA 80:6897–6901

    Article  PubMed  CAS  Google Scholar 

  • Davidson JN, Patterson D (1979) Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis. Proc Natl Acad Sci USA 76:1731–1735

    Article  PubMed  CAS  Google Scholar 

  • Davidson JN, Patterson D (1984) Genes encoding multifunctional proteins—the equivalent of bacterial operons. DNA 3:78

    Google Scholar 

  • Davidson JN, Carnright DV, Patterson D (1979) Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cell: III. Association of carbamyl phosphate synthtase, aspartate transcarbamoylase, and dihydroorotase in mutants of cultured Chinese hamster cells. Somatic Cell Genet 5:175–191

    Article  PubMed  CAS  Google Scholar 

  • Davidson JN, Rumsby PC, Tamaren J (1981) Organization of a multifunctional protein in pyrimidine biosynthesis. J Biol Chem 256:5220–5225

    PubMed  CAS  Google Scholar 

  • Evans DR (1986) CAD, a chimeric protein that initiatesde novo pyrimidine biosynthesis in higher eucaryotes. In: Hardie DG, Coggins JR (eds) Multidomain proteins—structure and evolution. Elsevier, Amsterdam, pp 283–331

    Google Scholar 

  • Felix H (1982) Permeabilized cells. Anal Biochem 120:211–234

    Article  PubMed  CAS  Google Scholar 

  • Foltermann KF, Shanley MS, Wild JR (1984) Assembly of the aspartate transcarbamoylase holoenzyme from transcriptionally independent catalytic and regulatory cistrons. J Bacteriol 157:891–898

    PubMed  CAS  Google Scholar 

  • Foltermann KF, Wild JR, Zink DL, O'Donovan GA (1981) Regulatory variance of aspartate transcarbamoylase among strains ofYersinia enterocolitica andYersinia enterocolitica like organisms. Curr Microbiol 6:43–47

    Article  CAS  Google Scholar 

  • Foltermann KF, Beck DA, Wild JR (1986)In vivo formation of hybrid aspartate transcarbamoylase from native subunits of divergent members of the familyEnterobacteriaceae. J Bacteriol 167:285–290

    PubMed  CAS  Google Scholar 

  • Freund JN, Jarry BP (1987) Therudimentary gene ofDrosophila melanogaster encodes four enzymic functions. J Mol Biol 193:1–13

    Article  PubMed  CAS  Google Scholar 

  • Gerhart JC, Schachman HK (1965) Distinct subunits for the regulation and catalytic activity of aspartate transcarbamoylase. Biochemistry 4:1054–1062

    Article  PubMed  CAS  Google Scholar 

  • Gilbert W, Marchionni M, McKnight G (1986) On the antiquity of introns. Nature 271:501

    Article  Google Scholar 

  • Grayson DR, Evans DR (1983) The isolation and characterization of the aspartate transcarbamoylase domain of the multifunctional protein, CAD. J Biol Chem 258:4123–4129

    PubMed  CAS  Google Scholar 

  • Grayson DR, Lee L, Evans DR (1985) Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells. J Biol Chem 260:15840–15849

    PubMed  CAS  Google Scholar 

  • Hoffman CS, Wright A (1985) Fusions of secreted proteins to alkaline phosphatase: an approach for studying secretion. Proc Natl Acad Sci USA 82:5107–5111

    Article  PubMed  CAS  Google Scholar 

  • Honzatko RB, Lipscomb WN (1982) Interactions of phosphate ligands ofEscherichia coli aspartate carbamoyltransferase in the crystalline state. J Mol Biol 160:264–286

    Google Scholar 

  • Honzatko RB, Crawford JL, Monaco HL, Ladner JE, Edwards BFP, Evans DR, Warren SF, Wiley DC, Ladner RC, Lipscomb WN (1982) Crystal and molecular structures of native and ATP-liganded aspartate carbamoyltransferase fromEscherichia coli. J Mol Biol 160:219–263

    Article  PubMed  CAS  Google Scholar 

  • Hoover TA, Roof WD, Foltermann KF, O'Donovan GA, Bencini DA, Wild JR (1983) Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase ofEscherichia coli. Proc Natl Acad Sci USA 80:2462–2466

    Article  PubMed  CAS  Google Scholar 

  • Houghton JE (1986) Structural and functional comparisons between OTCase and ATCase, leading to the formation and characterization of an active “domain fusion” betweenargl (OTCase) andpyrB (ATCase). Dissertation, Texas A&M University, College Station TX

    Google Scholar 

  • Jones ME (1980) Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Annu Rev Biochem 49:253–279

    Article  PubMed  CAS  Google Scholar 

  • Kania J, Mueller-Hill B (1980) Artificial protein chimeras. In: Bisswanger H, Schmincke-Ott E (eds) Multifunctional proteins. Wiley, New York, pp 31–47

    Google Scholar 

  • Kedzie KM (1987) Characterization of thepyrB gene ofSerratia marcescens and hybrid gene formation with thepyrB gene ofEscherichia coli, leading to the production of chimeric ATCases. Dissertation, Texas A&M University, College Station TX

    Google Scholar 

  • Keppler D, Holstege A (1982) Pyrimidine nucleotide metabolism and its compartmentation. In: Sies H (ed) Metabolic compartmentation. Academic Press, London, pp 149–203

    Google Scholar 

  • Krause KL, Volz KW, Lipscomb WN (1987) 2.5 A structure of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. J Mol Biol 193:527–553

    Article  PubMed  CAS  Google Scholar 

  • Lerner CG, Switzer RL (1986) Cloning and structure of theBacillus subtilis aspartate transcarbamoylase gene (pyrB). J Biol Chem 261:11156–11165

    PubMed  CAS  Google Scholar 

  • Lovatt CJ, Cheng AH (1984) Aspartate transcarbamoylase. Site of end product inhibition of the orotate pathway in intact cells ofCucurbita pepo. Plant Physiol 75:511–515

    Article  PubMed  CAS  Google Scholar 

  • Lue PF, Kaplan JG (1969) The aspartate transcarbamoylase and carbamoyl phosphate synthetase of yeast: a multifunctional enzyme complex. Biochem Biophys Res Commun 34:426–433

    Article  PubMed  CAS  Google Scholar 

  • Makoff AJ, Radford A (1978) Genetics and biochemistry of carbamoyl phosphate biosynthesis and its utilization in the pyrimidine biosynthetic pathway. Microbiol Rev 42:307–328

    PubMed  CAS  Google Scholar 

  • Maley JA, Davidson JN (1988) The aspartate transcarbamoylase domain of a mammalian multifunctional protein expressed as an independent enzyme inEscherichia coli. Mol Gen Genet 213:278–284

    Article  PubMed  CAS  Google Scholar 

  • Maley JA, Niswander LA, Flower AM, Davidson JN (1984) Dissecting the organization of the hamster CAD gene by expression of cDNA pieces inE. coli mutants. DNA 3:93

    Google Scholar 

  • Mally MI, Grayson DR, Evans DR (1981) Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells: evidence for discrete domains. Proc Natl Acad Sci USA 78:6647–6651

    Article  PubMed  CAS  Google Scholar 

  • Mas MT, Chen CY, Hitzeman RA, Riggs AD (1986) Active human-yeast chimeric phosphoglycerate kinases engineered by domain interchange. Science 233:788–790

    Article  PubMed  CAS  Google Scholar 

  • Messing JRC, Seeburg PH (1981) A system for shot-gun DNA sequencing. Nucleic Acids Res 9:309–321

    Article  PubMed  CAS  Google Scholar 

  • Monaco HL, Crawford JL, Lipscomb WN (1978) Three-dimensional structure of aspartate carbamyltransferase fromEscherichia coli and of its complex with cytidine triphosphate. Proc Natl Acad Sci USA 75:5276–5280

    Article  PubMed  CAS  Google Scholar 

  • Nishikawa K (1983) Assessment of secondary structure prediction of protein comparison of computerized Chou-Fasman method with others. Biochim Biophys Acta 748:285–299

    PubMed  CAS  Google Scholar 

  • Ong BL, Jackson JF (1972) Pyrimidine nucleotide biosynthesis inPhaseolus aureus. Biochem J 129:583–593

    PubMed  CAS  Google Scholar 

  • Prescott LM, Jones ME (1969) Modified methods for the determination of carbamyl phosphate. Analyt Biochem 32:408–419

    Article  PubMed  CAS  Google Scholar 

  • Rashin AA (1981) Location of domains in globular proteins. Nature 291:85–87

    Article  PubMed  CAS  Google Scholar 

  • Roof WD, Foltermann KF, Wild JR (1982) ThepyrB operon ofE. coli possesses arho independent attenuator sequence. Mol Gen Genet 187:391–400

    Article  PubMed  CAS  Google Scholar 

  • Ruiz JC, Wahl GM (1986)Escherichia coli aspartate transcarbamoylase: a novel marker for studies of gene amplification and expression in mammalian cells. Mol Cell Biol 6:3050–3058

    PubMed  CAS  Google Scholar 

  • Rumsby PC, Campbell PC, Niswander LA, Davidson JN (1984) Organization of a multifunctional protein in pyrimidine biosynthesis. Biochem J 217:435–440

    PubMed  CAS  Google Scholar 

  • Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693

    PubMed  CAS  Google Scholar 

  • Sanger FS, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Schneider WP, Nichols BP, Yanofsky C (1981) 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 USA 78:2169–2173

    Article  PubMed  CAS  Google Scholar 

  • Shigesada K, Stark GR, Maley JA, Niswander LA, Davidson JN (1985) Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamoylase. Mol Cell Biol 5:1735–1742

    PubMed  CAS  Google Scholar 

  • Silhavy TS, Berman ML, Enquist LW (eds) (1984) Experiments with gene fusions. Cold Spring Harbor Press, Cold Spring Harbor NY, pp 273–281

    Google Scholar 

  • Simon LD, Randolph B, Irwin N, Binkowski G (1983) Stabilization of proteins by a bacteriophage T4 gene cloned inEscherichia coli. Proc Natl Acad Sci USA 80:2059–2062

    Article  PubMed  CAS  Google Scholar 

  • Stark GS (1977) Multifunctional proteins: one gene—more than one enzyme. Trends Biochem Sci 2:64–66

    Article  CAS  Google Scholar 

  • Swamy KHS, Goldberg AL (1981)E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature 292:652–654

    Article  PubMed  CAS  Google Scholar 

  • Traut TW (1986) Are proteins made of modules? Mol Cell Biochem 70:3–10

    Article  PubMed  CAS  Google Scholar 

  • Volz KW, Krause KL, Lipscomb WN (1986) The binding of N-(phosphonacetyl)-L-aspartate to aspartate carbamoyltransferase ofEscherichia coli. Biochem Biophys Res Commun 136:822–836

    Article  PubMed  CAS  Google Scholar 

  • Wild JR, Foltermann KF, O'Donovan GA (1980) Regulatory divergence of aspartate transcarbamoylases within the Enterobacteriaceae. Arch Biochem Biophys 201:506–517

    Article  PubMed  CAS  Google Scholar 

  • William LG, Bernhardt S, Davis RH (1970) Copurification of pyrimidine specific carbamyl phosphate synthetase and aspartate transcarbamylase ofNeurospora crassa. Biochemistry 9:4329–4335

    Article  Google Scholar 

  • Zinder ND, Boeke JD (1982) The filamentous phage as vectors for recombinant DNA—a review. Gene 19:1–10

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. John E. Houghton

    Present address: Department of Biology, Yale University, 06511, New Haven, Connecticut, USA

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, 77843, College Station, Texas, USA

    Joseph G. Major Jr., Melinda E. Wales, John E. Houghton & James R. Wild

  2. Department of Microbiology and Immunology, University of Kentucky, Albert B. Chandler Medical Center, 40536, Lexington, Kentucky, USA

    Julie A. Maley & Jeffrey N. Davidson

Authors
  1. Joseph G. Major Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Melinda E. Wales
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John E. Houghton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julie A. Maley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey N. Davidson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James R. Wild
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Major, J.G., Wales, M.E., Houghton, J.E. et al. Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase. J Mol Evol 28, 442–450 (1989). https://doi.org/10.1007/BF02603079

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02603079

Key words

Search

Navigation