Molecular analysis of an aromatic degradative pathway: Studies on the genes and enzymes for homoprotocatechuate degradation from Escherichia coli C.

摘要

The homoprotocatechuate degradative pathway of Escherichia coli C contains two isomerisation reactions with chemically similar intermediates, which were thought to be catalysed by distinct but genetically linked enzymes. The possibility that these two isomerases may have arisen from the same ancestral precursor, given their similar substrate structures and close physical location of their genes, was investigated by nucleotide sequencing and purification of the individual enzymes. The purified proteins are of very different subunit molecular weight and have been shown by kinetic measurements to be specific for their respective substrates. The two isomerisation events are separated by an enzyme catalysed decarboxylation and it has been shown that the second isomerisation and the decarboxylation reactions are distinct activities of the same protein subunit. Comparison of the amino acid sequence of the latter with that of the first isomerase of the pathway, reveals a very low level of similarity, suggesting that the two enzymes are unlikely to be derived from a common ancestor. Subcloning of the rest of hpc gene cluster has revealed that the gene order and direction of transcription are not as previously reported. All the genes for the pathway enzymes are transcribed in the same direction and are subject to negative regulation by a protein which appears to be transcribed in the opposite direction. A putative operator site to which the regulatory protein could bind has been located in the same region as a mapped promoter for the pathway genes, which also contains a binding site for the catabolite activator protein. Pairwise comparison of the amino acid sequences of the rest of the Hpc enzymes have shown only low levels of similarity. However, the single dehydrogenase enzyme of the pathway is similar to isoforms of human aldehyde dehydrogenase. The subcloning procedures used in this study have enabled high level expression of several of the pathway enzymes. This has enabled preliminary crystallographic analysis of the isomerase and decarboxylase/isomerase enzymes.