Thibaut D, Ratet N, Bisch D, Faucher D, Debussche L, Blanche F
Département Analyse, Centre de Recherche de Vitry-Alfortville, Rhône-Poulenc Rorer S.A., Vitry-sur-Seine, France.
J Bacteriol. 1995 Sep;177(18):5199-205. doi: 10.1128/jb.177.18.5199-5205.1995.
High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.
在螺旋链霉菌及其他一些链阳性菌素A产生菌体内，可实现较高水平的14C标记的原始霉素IIB（PIIB）向原始霉素IIA（PIIA）的转化。这证实了PIIB是通往PIIA途径中的一个中间体。此外，对无细胞蛋白质制剂的体外研究表明，PIIB氧化为PIIA是一个复杂的过程，需要NADH、核黄素5'-磷酸（FMN）和分子氧。已证明催化该反应需要两种酶。通过基于PIIA形成的偶联酶测定法，并通过添加互补酶，从螺旋链霉菌中纯化得到了这两种均具有同质性的酶。其中一种酶通过包括在FMN-琼脂糖上进行关键亲和色谱步骤的方法纯化了约3000倍。结果表明它是一种NADH：FMN氧化还原酶（E.C. 1.6.8.1.）（以下称为FMN还原酶），为更丰富的第二种酶提供还原型FMN（FMNH2）。后者仅纯化了160倍，被称为PIIA合酶。我们的数据有力地表明，该酶催化PIIB被分子氧瞬时羟基化，随后立即脱水生成PIIA。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，天然PIIA合酶由两个不同的亚基组成，分子量约为50,000和35,000，而FMN还原酶似乎是一种单体，分子量约为28,000，含有一分子紧密结合的FMN。对整个多肽或其一些经胰蛋白酶消化的片段进行逐步的埃德曼降解，得到了两种分离蛋白的氨基酸序列。
