Rodphaya D, Nihira T, Yamada Y
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
J Antibiot (Tokyo). 1989 May;42(5):752-60. doi: 10.7164/antibiotics.42.752.
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.
帕特洛利德是由荨麻青霉S11R59产生的一组12元大环内酯类抗生素。从荨麻青霉S11R59中纯化并鉴定了一种参与将帕特洛利德C转化为帕特洛利德A的酶。该酶在SDS-PAGE和分子筛HPLC上均显示为单一条带，两者均表明其分子量为86,000，表明该酶是单体。然而，通过等电聚焦，该酶被分离为两条pI非常相似（pI 4.2和4.3）的条带。活性染色表明，两条带均催化帕特洛利德C向帕特洛利德A的转化。在将帕特洛利德C转化为帕特洛利德A的过程中同时产生H2O2，证明这两种同工酶是氧化酶。确定帕特洛利德C、A和H2O2的摩尔比为1:1:1。确定最佳pH和温度分别为7和35-40℃，并且该酶在pH 6-9和4-40℃下稳定。氧化酶在345和450 nm处显示出特征吸收，表明存在黄素作为辅酶。在所测试的几种帕特洛利德C类似物中，氧化酶显示出非常窄的底物特异性；只有帕特洛利德C被氧化为帕特洛利德A。荨麻青霉S11R59的无细胞提取物中不存在从帕特洛利德A到帕特洛利德C的逆反应的酶活性。因此，帕特洛利德C氧化酶在帕特洛利德的生物合成中起关键作用。
