Purification and characterization of 3-dehydroshikimate dehydratase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.
3-Dehydroshikimate dehydratase catalyzes the third reaction in the inducible quinic acid catabolic pathway of Neurospora crassa and is encoded in the qa-4 gene of the qa gene cluster. As part of continuing genetic and biochemical studies concerning the organization and regulation of this gene cluster, 3-dehydroshikimate dehydratase has been purified and characterized biochemically. The enzyme was purified 1650-fold using the following techniques: 1) (NH4)2SO4 fractionation; 2) ion exchange chromatography on DEAE-cellulose; 3) gel filtration on Sephadex G-100; 4) ion exchange chromatography on Cellex QAE (quaternary aminoethyl); and 5) hydroxylapatite chromatography. 3-Dehydroshikimate dehydratase is a monomer with a molecular weight of about 37,000 and a sedimentation coefficient of 3.27 S. It has a Km value of 5.9 X 10(-4) and an average isoelectric point of 4.92. The purified enzyme is extremely sensitive to thermal denaturation but can be significantly stabilized by Mg2+ ions. The purified enzyme also exhibits maximal catalytic activity only when assayed in the presence of certain divalent cations, e.g. magnesium. The NH2-terminal residue of 3-dehydroshikimate dehydratase is proline, and its alpha-amino group is unblocked.
D000596:Amino Acids / Q000032:analysis; D004355:Drug Stability; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D012765:Shikimic Acid / Q000031:analogs & derivatives
D000596:Amino Acids; D012765:Shikimic Acid; D006836:Hydro-Lyases