Oxidases involved in biosynthesis of macrolide antibiotic patulolides from Penicillium urticae S11R59.
Patulolides are a group of 12-membered macrolide antibiotics produced by Penicillium urticae S11R59. An enzyme involved in the conversion of patulolide C to patulolide A was purified from P. urticae S11R59 and characterized. The enzyme showed a single band on SDS-PAGE and molecular sieve HPLC both of which indicated a Mr of 86,000, indicating that the enzyme is monomeric. However, the enzyme was separated into two bands of very similar pI's (pI 4.2 and 4.3) by isoelectric focusing. Both bands catalyzed the conversion of patulolide C to patulolide A, as demonstrated by activity staining. The two isoenzymes were proved to be oxidases by the simultaneous production of H2O2 during the conversion of patulolide C to patulolide A. The molar ratio for patulolides C, A and H2O2 was determined to be 1:1:1. The optimum pH and temperature were determined to be 7 and 35-40 degrees C, respectively, and the enzymes were stable at pH 6-9 and 4-40 degrees C. The oxidases showed characteristic absorption at 345 and 450 nm, indicating the presence of flavin as coenzyme. Among several analogues of patulolide C tested, the oxidases showed very narrow substrate-specificity; only patulolide C was oxidized to patulolide A. No enzyme activity for the reverse reaction, i.e. from patulolide A to patulolide C, was present in the cell-free extract of P. urticae S11R59. Patulolide C oxidases therefore play a key role in the biosynthesis of patulolides.
Rodphaya
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D
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Nihira
T
T
Yamada
Y
Y
D000935:Antifungal Agents / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000378:metabolism; D007783:Lactones / Q000096:biosynthesis / Q000378:metabolism; D018942:Macrolides; D015394:Molecular Structure; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
D000935:Antifungal Agents; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; C049727:patulolide C; C049725:patulolide A; D006861:Hydrogen Peroxide; D010088:Oxidoreductases