Purification, characterization and functional cloning of inositol oxygenase from Cryptococcus.
The enzyme inositol oxygenase (myo-inositol : oxygen oxidoreductase; E.C. 1.13.99.1) is a monooxygenase that converts inositol into glucuronic acid in the presence of molecular oxygen. This enzyme is integrated into a pathway leading to either degradation and energy production or the biosynthesis of precursors for polysaccharides. The enzyme was purified from the yeast Cryptococcus lactativorus by a five-step chromatography procedure. The purified enzyme shows a molecular mass of 37 kDa on SDS-PAGE, similar to the estimation of the size of the native enzyme determined by size exclusion chromatography. Peptides of the inositol oxygenase protein derived from a tryptic digest were sequenced de novo by nanoelectrospray tandem mass spectrometry. Using degenerate oligonucleotides, the corresponding gene was cloned from first strand cDNA. The open reading frame encodes a 315 amino acid polypeptide with a predicted molecular mass of 36.9 kDa. Inositol oxygenase is a single copy gene in C. lactativorus. It has close homologues in other fungi such as Cryptococcus neoformans and Neurospora crassa. Biochemical characterization of the enzyme showed a pH optimum of 6-6.5 and a temperature optimum of 30 degrees C. Myo-inositol is the only accepted substrate with a Km of ca. 5 mM. The enzyme contains a Fe-centre but the enzyme activity is resistant to KCN.
Kanter
Ulrike
U
University of Frankfurt, Biocentre, Plant Molecular Biology, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
Becker
Michael
M
Friauf
Eckhard
E
Tenhaken
Raimund
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology* / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D050562:Inositol Oxygenase; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization
D004271:DNA, Fungal; D010105:Oxygenases; D050562:Inositol Oxygenase