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Appl Environ Microbiol. 1992 September; 58(9): 2792-2798

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.

Department of Microbiology and Cell Science, University of Florida, Gainesville 32611-0100.

ABSTRACT

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.(ABSTRACT TRUNCATED AT 250 WORDS)

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