Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans.
The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the overproduced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.
Dept. of Biochemistry and Genetics, University of Newcastle upon Tyne, UK.
J D
JD
Moore
A R
AR
Hawkins
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D001233:Aspergillus nidulans / Q000201:enzymology*; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000235:genetics; D008190:Lyases / Q000096:biosynthesis* / Q000235:genetics; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor); D011994:Recombinant Proteins / Q000096:biosynthesis; D014166:Transferases
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C019728:arom enzyme; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase; D014166:Transferases; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D008190:Lyases; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase