E. coli p-aminobenzoate synthetase and anthranilate synthetase are structurally and genetically related enzymes that catalyze similar, but slightly different, reactions. Each enzyme is composed of two dissimilar subunits, one responsible for glutamine amidotransferase (GAT) activity, and the other responsible for the aromatization of chorismate. The difference in the products is the disposition of amino- and carboxy- substituents on a benzene ring. The subunits of PABS are encoded by pabA and pabB and the subunits of anthranilate synthetase are encoded by trpE and trpG. The nucleotide and amino acid sequences of these four genes have been determined, and clearly indicate divergence from a common ancestor. Similarity is approximately 30% between the large subunits and 45% between the small subunits of the enzymes. The GAT subunits perform identical functions, and differ only in the specific interactions with the appropriate large subunit. The nucleotide sequence of a GAT subunit that functions in both anthranilate and PABA synthesis in A. calcoaceticus has also been determined, and is very similar to the E. coli pabA sequence. The experiments in this proposal focus on the determination of specific differences between the pab and trp genes that are responsible for divergence of function. The experimental approaches include construction of hybrid genes by exploiting in vivo recombination and in vitro DNA replication techniques, in vitro inter- and intrageneric subunit exchange, in vitro mutagenesis, and analysis of second site reversions. The results of the proposed studies will include information on the ways in which genes evolve from a common ancestor after a gene duplication event. The extent of total nucleotide and amino acid sequence divergence has been documented, and my aim is to determine the fraction of the divergence that is responsible for alteration of function. We will also gain insight into a mechanism in which biochemical specificity has been maintained while function has diverged. The approach and methodology are unique in that a functional determination of the consequences of sequence divergence will accompany a descriptive approach on the extent of sequence divergence.
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