Loss of allosteric control but retention of the bifunctional catalytic competence of a fusion protein formed by excision of 260 base pairs from the 3' terminus of pheA from Erwinia herbicola

• Published in: Applied and Environmental Microbiology 1992-09, Vol.58 (9), p.2792-2798
• Main Authors: Xia, T. (University of Florida, Gainesville, FL), Zhao, G, Jensen, R.A
• Format: Article
• Language: English
• Subjects: ACTIVIDAD CATALITICA; ACTIVIDAD ENZIMATICA; ACTIVITE CATALYTIQUE; ACTIVITE ENZYMATIQUE; Allosteric Regulation - genetics; Allosteric Regulation - physiology; Amino Acid Sequence; Bacterial Proteins - genetics; Bacteriology; Base Sequence; Catalysis; Chorismate Mutase - genetics; Cloning, Molecular; ERWINIA; Erwinia - enzymology; Erwinia - genetics; Erwinia herbicola; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Genetic Vectors; Genetics; Molecular Sequence Data; Multienzyme Complexes; NUCLEOTIDE; NUCLEOTIDOS; Phenylalanine - genetics; Prephenate Dehydratase - genetics; PROTEINAS; PROTEINE; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sequence Homology
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.58.9.2792-2798.1992

A bifunctional protein denoted as the P protein and encoded by pheA is widely present in purple gram-negative bacteria. This P protein carries catalytic domains that specify chorismate mutase (CM-P) and prephenate dehydratase. The instability of a recombinant plasmid carrying a pheA insert cloned from Erwinia herbicola resulted in a loss of 260 bp plus the TAA stop codon from the 3' terminus of pheA. The plasmid carrying the truncated pheA gene (denoted pheA ) was able to complement an Escherichia coli pheA auxotroph. pheA was shown to be a chimera composed of the residual 5' part of pheA (901 bp) and a 5-bp fragment from the pUC18 vector. The new fusion protein (PheA ) retained both chorismate mutase and prephenate dehydratase activities. PheA had a calculated subunit molecular weight of 33,574, in comparison to the 43,182-molecular-weight subunit size of PheA. The deletion did not affect the ability of PheA to assume the native dimeric configuration of PheA. Both the CM-P and prephenate dehydratase components of PheA were insensitive to L-phenylalanine inhibition, in contrast to the corresponding components of PheA. L-phenylalanine protected both catalytic activities of PheA from thermal inactivation, and this protective effect of L-phenylalanine upon the PheA activities was lost. PheA was more stable than PheA to thermal inactivation; this was more pronounced for prephenate dehydratase than for CM-P. In the presence of dithiothreitol, the differential resistance of PheA prephenate dehydratase to thermal inactivation was particularly striking. We conclude that the deletion defines a discrete regulatory domain for allosteric control of pheA and that this domain is located on the carboxy terminus of PheA and is separable from the two catalytic domains. Excision of the regulatory domain not only abolished allosteric control but secondarily influenced catalytic and physical properties