title: Characterization of the 3-dehydroquinase domain of the pentafunctional AROM protein, and the quinate dehydrogenase from Aspergillus nidulans, and the overproduction of the type II 3-dehydroquinase from neurospora crassa.
abstract: The AROM protein of Aspergillus nidulans is a multidomain pentafunctional polypeptide that is active as a dimer and catalyses steps 2-6 in the prechorismate section of the shikimate pathway. The three C-terminal domains (including the type I 3-dehydroquinase) of the AROM protein are homologous with the qutR-encoded QUTR protein that represses transcription of the eight genes comprising the quinic acid utilization (qut) gene cluster, and the two N-terminal domains are homologous with the qutA-encoded QUTA protein that transcribes the qut genes. As part of a larger research programme designed to compare the structures of the three proteins and to probe the domain structure and interaction within each protein, we have overproduced and purified the 3-dehydroquinase domain of the AROM protein. Additionally we have overproduced and purified the qutB-encoded quinate dehydrogenase and overproduced the qa-2 encoded type II 3-dehydroquinase of Neurospora crassa. We report that the AROM 3-dehydroquinase domain has a monomeric native state, with an apparent kcat./Km ratio that is approx. 160-fold lower than the value for the native N. crassa AROM protein. The AROM protein 3-dehydroquinase domain is sensitive to inactivation by borohydride in the presence of the substrate 3-dehydroquinate, confirming that it is a typical type I 3-dehydroquinase. The purified quinate dehydrogenase is bifunctional, being able to metabolize shikimate as a substrate. The apparent Km values for quinate (450 microM), shikimate (1.7 mM) and NAD+ (150 microM) are all similar to values reported for the qa-3-encoded enzyme from N. crassa.
authors.affiliation: Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, U.K.
authors.forename: A R
authors.initials: AR
authors.lastname: Hawkins
authors.forename: J D
authors.initials: JD
authors.lastname: Moore
authors.forename: A M
authors.initials: AM
authors.lastname: Adeokun
mesh_terms: D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014166:Transferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C019728:arom enzyme; D000429:Alcohol Oxidoreductases; C022511:quinate dehydrogenase; D014166:Transferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D008190:Lyases; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase