Stegmann Evi, Pelzer Stefan, Bischoff Daniel, Puk Oliver, Stockert Sigrid, Butz Diane, Zerbe Katja, Robinson John, Süssmuth Roderich D, Wohlleben Wolfgang
Eberhard-Karls-Universität Tübingen, Fakultät Biologie, Mikrobiologisches Institut, Mikrobiologie/Biotechnologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
J Biotechnol. 2006 Aug 5;124(4):640-53. doi: 10.1016/j.jbiotec.2006.04.009. Epub 2006 May 30.
In the balhimycin biosynthesis three oxygenases OxyA, OxyB and OxyC are responsible for the oxidative phenol coupling reactions, which lead to the ring-closures between the aromatic amino acid side chains in the heptapeptide aglycone. These ring-closures constrain the peptide backbone into the cup-shaped conformation that is required for binding to the Lys-D-Ala-D-Ala-terminus of the cell wall precursor peptide and represent one of the essential features of glycopeptide antibiotics. In the balhimycin biosynthetic gene cluster the oxygenase genes oxyA, oxyB and oxyC have been identified downstream of the peptide synthetase genes. Reverse transcription (RT)-PCR analyses revealed that these oxygenase genes in Amycolatopsis balhimycina are co-transcribed. Non-polar mutants (NPoxyA, DeltaoxyB and DeltaoxyC) were constructed, cultivated in production medium and assayed for the presence of glycopeptides and glycopeptide precursors by HPLC-ESI-MS. The mutant NPoxyA produces mainly monocyclic, the mutant DeltaoxyB linear and the mutant DeltaoxyC bicyclic peptides. These results definitely confirm the sequence of the three oxidative ring-closing steps (OxyB-OxyA-OxyC). The heterologous complementation of the mutant strains with the corresponding oxygenase genes from the vancomycin producer A. orientalis restored the production of balhimycin, which proves the functional equivalence of the oxygenases from the balhimycin and vancomycin producer. For the first time it is now possible to combine the genetic data obtained from the balhimycin producer with the biochemical and structural data obtained from the vancomycin producer.
在巴龙霉素生物合成过程中，三种加氧酶OxyA、OxyB和OxyC负责氧化苯酚偶联反应，该反应导致七肽苷元中芳香族氨基酸侧链之间的环化。这些环化反应将肽主链约束成杯状构象，这是与细胞壁前体肽的Lys-D-Ala-D-Ala末端结合所必需的，并且代表了糖肽抗生素的基本特征之一。在巴龙霉素生物合成基因簇中，加氧酶基因oxyA、oxyB和oxyC已在肽合成酶基因的下游被鉴定出来。逆转录（RT）-PCR分析表明，这些加氧酶基因在巴龙霉素链霉菌中是共转录的。构建了非极性突变体（NPoxyA、DeltaoxyB和DeltaoxyC），在生产培养基中培养，并通过HPLC-ESI-MS检测糖肽和糖肽前体的存在。突变体NPoxyA主要产生单环肽，突变体DeltaoxyB产生线性肽，突变体DeltaoxyC产生双环肽。这些结果明确证实了三个氧化环化步骤的顺序（OxyB-OxyA-OxyC）。用来自万古霉素产生菌东方拟无枝酸菌的相应加氧酶基因对突变菌株进行异源互补，恢复了巴龙霉素的产生，这证明了来自巴龙霉素产生菌和万古霉素产生菌的加氧酶在功能上是等效的。现在首次有可能将从巴龙霉素产生菌获得的遗传数据与从万古霉素产生菌获得的生化和结构数据结合起来。
