Studies on the Shikimate Dehydrogenase Gene of Escherichia coli

Anton, Ian Alexander (1985) Studies on the Shikimate Dehydrogenase Gene of Escherichia coli. PhD thesis, University of Glasgow.

Abstract

Aromatic compounds are made via the shikimate pathway. The N. crassa pentafunctional arom enzyme has five shikimate pathway activities on one polypeptide whereas in E. coli all seven activities are separate enzymes. It has been hypothesised that arom arose by the fusion of genes for monofunctional enzymes. To test this proposal requires comparison of the sequences of arom and its monofunctional counterparts. Towards this future goal the author set out to sequence the E. coli aroE gene encoding shikimate dehydrogenase ("E3"). AroE was cloned from the previously isolated transducing phage spcl by selection in an aroE auxotroph. The E3 overexpressed by strains carrying these clones is identical to wild-type E3 by native and SDS PAGE. A protocol was developed which permits the renaturation of E3 after SDS PAGE. A 1.82 kbp region of DNA containing aroE was sequenced on both strands by the M13/dideoxy method. The open reading frame (ORF) corresponding to aroE was initially identified by size and by further subcloning. A high level of E3 overproduction was obtained by placing the aroE gene in an expression vector. This gave E3 specific activities more than 300 times higher than in wild-type cells. Overproduced E3 was purified to homogeneity using a previously developed method. 20g (vet weight) of cells yielded 10 mg of E3. The E3 amino acid sequence deduced from the DNA sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced E3. Within the 1.82 kbp of DNA sequenced on both strands, together with the adjacent 0. 6 kbp sequenced on only one strand, two large ORF's were found in addition to aroE ("UPSORF 1" and "UPSORF 2"). Biased codon utilisation and Fickett analysis hinted that UPSORF's 1 and 2 might be genes, as might be a truncated ORF ("UPSORF 3?") at the end of the single strand sequence. It remains to be seen if these UPSORF's encode proteins. Strong indirect evidence that UPSORF 2 encodes a protein comes from its predicted amino acid sequence (180 a.a.'s) which contains four internal homologous sub-regions, suggesting internal gene duplication. In particular, each sub-region has two pairs of cysteine residues. Preliminary sequence comparisons indicate the possibility of very weak homologies between UPSORF 2 and some iron-sulphur proteins. Truncation of sequences ≥1.4 kbp and ≥0.8 kbp upstream from the 5' end of aroE may be the cause of the observed 4-5 fold and 20-23 fold reductions (respectively) in E3 specific activity with some of the smaller aroE subclones (relative to the larger subclones). This may be indirect evidence that aroE is part of a "mixed" operon, with UPSORF's 1, 2, and 3, analogous to that found for aroA.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Genetics
• Date of Award: 1985
• Depositing User: Enlighten Team
• Unique ID: glathesis:1985-76585
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 14:06
• Last Modified: 19 Nov 2019 14:06
• URI: https://theses.gla.ac.uk/id/eprint/76585