Over-expression of the yeast multifunctional arom protein.
The pentafunctional arom protein of Saccharomyces cerevisiae is encoded by the ARO1 gene. Substantial elevation of the levels of the arom protein (25-fold) was achieved in yeast using a vector that exploited the ubiquitin-fusion cleavage system of yeast. However, attempts to express the N-terminal 3-dehydroquinate synthase domain (E1) or the internal 3-dehydroquinase domain (E2) using the same system did not succeed. The yeast arom protein was successfully purified from the over-expressing transformant, and was found to possess all five enzymatic activities in a ratio similar to that observed in crude cell extracts. The purified material consisted mainly of a polypeptide that co-migrated in SDS-PAGE with intact arom proteins from other species.
Graham
L D
LD
Department of Biochemistry, University of Glasgow, Scotland, UK.
Gillies
F M
FM
Coggins
J R
JR
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019761:Phosphorus-Oxygen Lyases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014166:Transferases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C019728:arom enzyme; D017886:Durapatite; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase; D014166:Transferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D008190:Lyases; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases