From Biological Diversity to Structure-Function Analysis: Protein Engineering in Aspartate Transcarbamoylase
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.
The catalytic polypeptides of various ATCases are organized into two discrete and separable binding domains for its substrates, carbamoyl phosphate and aspartate.
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.
There are sub-domain structural units within the various polypeptides which have a coordinated impact on specific catalytic and regulatory functions in the enzyme.
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, 184.108.40.206), 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.
KeywordsRegulatory Subunit Pyrimidine Biosynthesis Chimeric Enzyme Zinc Binding Domain Hybrid Enzyme
Unable to display preview. Download preview PDF.
- 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.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
- 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
- 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
- 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
- 23.S.T. Berth and D.R. Evans. Characterization of the aspartate transcarbamoylase of Pseudomonas fluorescens. FASEB J. 4A1836.Google Scholar
- 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
- 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.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
- 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.K.M. Kedzie, GA. O’Donovan and J.R. Wild. In preparation.Google Scholar
- 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.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
- 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.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
- 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
- 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.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