Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis.
The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable, is active as a homododecamer and has the lowest reported Km value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA- qutB- double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE- mutant strain of A. nidulans to utilize quinate as sole carbon source.
Moore
J D
JD
Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K.
Lamb
H K
HK
Garbe
T
T
Servos
S
S
Dougan
G
G
Charles
I G
IG
Hawkins
A R
AR
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D001233:Aspergillus nidulans / Q000201:enzymology*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005800:Genes, Fungal; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification; D008190:Lyases / Q000235:genetics*; D009097:Multienzyme Complexes / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010770:Phosphotransferases / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011401:Promoter Regions, Genetic; D011801:Quinic Acid / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis; D012485:Salmonella typhi / Q000201:enzymology*; D014166:Transferases
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C019728:arom enzyme; D011801:Quinic Acid; D000429:Alcohol Oxidoreductases; D014166:Transferases; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D008190:Lyases; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase