Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase |
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Authors: | Joseph G Major Jr Melinda E Wales John E Houghton Julie A Maley Jeffrey N Davidson James R Wild |
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Institution: | (1) Department of Biochemistry and Biophysics, Texas A&M University, 77843 College Station, Texas, USA;(2) Department of Microbiology and Immunology, University of Kentucky, Albert B. Chandler Medical Center, 40536 Lexington, Kentucky, USA;(3) Present address: Department of Biology, Yale University, 06511 New Haven, Connecticut, USA |
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Abstract: | 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. |
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Keywords: | Gene fusion Protein evolution Multifunctional enzyme complexes |
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