Studies on the aroB, aroC and aroL genes of Escherichia coli

The arom pentafunctional enzyme found in fungi catalyses five consecutive reactions of the shikimate pathway. The same aromatic biosynthetic functions in bacteria are achieved by five monofunctional enzymes, products of individual unlinked genes. The evolutionary implications and questions raised by these two extremes of structural organisation can be addressed by comparison of the primary structures of the five monofunctional activities with that of the multifunctional enzyme. This thesis is concerned with contributing some of the required bacterial data. The complete amino acid sequence of the Escherichia coli 3-dehydroquinate synthase (aroB gene product) has been determined by a combined nucleotide and direct amino acid sequencing strategy. The aroB gene was sub-cloned from the plasmid pJB14, its nucleotide sequence was determined and an overexpressing (inducible) strain constructed. The gene product DHQ synthase was purified from this strain and its N-terminal amino acid sequence determined. E. coli DHQ synthase is 362 amino acids long with a calculated of 38,880. Analysis of the aroB nucleotide sequence and its 5' and 3' flanking regions has identified the aroB promoter and possible 3' terminator sequences. The aroL gene encoding the tyrR regulated shikimate kinase II was cloned from E. coli K12. The aroL gene has been identified by nucleotide sequencing and direct N-terminal amino acid sequencing. Construction of overexpressing strains has allowed purification (for the first time) of a monofunctional shikimate kinase. E. coli shikimate kinase II is monomeric with a calculated of 18,937. The amino acid sequence contains a region with some homology to sequences found in other kinases and ATP-requiring enzymes. Transcript mapping has identified a possible operator sequence overlapping the aroL promoter which could constitute the tyrR repressor binding site. The aroC gene encoding chorismate synthase has been cloned from E. coli. The construction of overexpressing strains has allowed purification for the first time of E. coli chorismate synthase. The aroC gene has been identified by nucleotide sequencing and confirmed by N-terminal amino acid sequencing of the purified protein. E. coli chorismate synthase is 357 amino acids long with a calculated of 38,183. Analysis of the 3' flanking sequences has identified possible terminator elements. The bacterial sequences for the aroB and aroL gene products have been compared with the S. cerevisiae and A. nidulans arom amino acid sequences. Discrete non-overlapping regions of the fungal polypeptide are homologous with both E. coli DHQ synthase (aroB), shikimate kinase (aroL) and the remaining aroA, aroD and aroE gene products. This evidence supports the hypothesis which forms the basis of this study: that the fungal arom multifunctional enzyme is a mosaic of five independently folded 'domains'