Domain structure and interaction within the pentafunctional arom polypeptide.
The AROM locus of Aspergillus nidulans specifies a pentafunctional polypeptide catalysing five consecutive steps leading to the production of 5-enolpyruvylshikimate 3-phosphate in the shikimate pathway. Aided by oligonucleotide-mediated site-directed mutagenesis, the whole AROM locus and various overlapping subfragments from within it have been fused to the powerful hybrid trc promoter in the Escherichia coli plasmid pKK233-2. Expression of these subfragments in appropriate aro mutants of E. coli has (a) allowed the delineation of functional domains within the arom polypeptide, (b) shown that the arom polypeptide falls in two independently folding and functioning regions, the N-terminal half specifying 3-dehydroquinate (DHQ) synthase and EPSP synthase and the C-terminus specifying shikimate kinase, biosynthetic 3-dehydroquinase (DHQase) and shikimate dehydrogenase, and (c) strongly suggested an interaction between the DHQ synthase and EPSP synthase domains to stabilise the EPSP synthase activity. In addition an isoenzyme of biosynthetic DHQase, catabolic DHQase, encoded by the QUTE gene of A. nidulans has been transcribed from the trc promoter and upon isopropyl-thio-beta-D-galactoside induction produces up to 20% of the total soluble cell protein.
Hawkins
A R
AR
Dept. of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, England.
Smith
M
M
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D019883:Alkyl and Aryl Transferases; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics*; D008190:Lyases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics*; D016296:Mutagenesis; D019761:Phosphorus-Oxygen Lyases; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D014166:Transferases / Q000032:analysis
D009097:Multienzyme Complexes; C019728:arom enzyme; D000429:Alcohol Oxidoreductases; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D008190:Lyases; D006836:Hydro-Lyases; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases