An unexpected interaction between the modular polyketide synthases, erythromycin DEBS1 and pikromycin PikAIV, leads to efficient triketide lactone synthesis
An unexpected interaction between the modular polyketide synthases, erythromycin DEBS1 and pikromycin PikAIV, leads to efficient triketide lactone synthesis
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DOI：
10.1021/bi0256779
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发表时间：
2002-09-03
期刊：
影响因子：
2.9
通讯作者：
Reynolds, KA
中科院分区：
文献类型：
--
作者：
Kim, BS;Cropp, TA;Reynolds, KA
金黄色葡萄球菌6-模吡克罗霉素聚酮合成酶(PikpKS，PikA1-PikAIV)的一个不同寻常的特征是能够产生12元和14元环大环内酯类化合物。含有最后一个延伸模块和硫代酯酶结构域的PikAIV组件负责产生这两种产物。在12元环大环内酯的情况下，PikAIII上的酰基酶中间体能够有效地“跳过”最后的延伸步骤，并被PikAIV的TE结构域环化，这可能是PikAIII-PikAIV相互作用的结果。在此，我们报道了DEBS1基因在金黄色葡萄球菌中的高效表达(PBK3)，该基因包括红霉菌6-脱氧赤藓内酯B合成酶的载体区和前两个模块，可高效生产153 mg/L的三酮内酯产物(TKLS)。表达DEBS1融合到TE结构域(DEBS1-TE)的质粒(PBK1)也观察到了类似水平的TKL。这些结果与之前的体内和体外分析形成了鲜明对比，在体内和体外分析中，只有DEBS1-TE有效地产生TKL。在pIkAIV和pILMIII-pIkAIV缺失宿主(0.5 mg/L)中，随着DEBS1基因的表达，TKLS水平显著降低，但不表达DEBS1-TE，加入pIkAIV互补质粒可部分恢复TKL的表达。这些数据表明，PikAIV能够有效地催化DEBS1上的酰基结合中间体形成6元内酯环产物，其方式与PikAIII的12元大环内酯产物相似。PikAIII和DEBS1的C-末端连接区的序列相似性和长度表明该区域可能负责与PiRAIV的相互作用。DEBS1的这一连接区被Pikai的相应区域替换后，在金黄色葡萄球菌中TKL的水平下降了95%，这与这一假设一致。
An unusual feature of the 6-module pikromycin polyketide synthase (PikPKS, PikAl-PikAIV) of S. venezuelae is the ability to generate both 12- and 14-membered ring macrolides. The PikAIV component containing the last extension module and a thioesterase domain is responsible for generating both of these products. In the case of the 12-membered ring macrolide, an acyl-enzyme intermediate on PikAIII is able to efficiently "skip" the last extension step and is cyclized by the TE domain of PikAIV, presumably as a result of a PikAIII-PikAIV interaction. Herein we report that plasmid-based expression (pBK3) of DEBS1, which comprises the loading domain and the first two modules of the Saccharopolyspora erythrea 6-deoxyerythronolide B synthase, in S. venezuelae leads to efficient 15 3 mg/L production of triketide lactone products (TKLs). Comparable levels of TKLs were observed with a plasmid (pBK1) which expressed DEBS1 fused to a TE domain (DEBS1-TE). These results are in stark contrast to previous in vivo and in vitro analyses, where only DEBS1-TE efficiently produces TKLs. Levels of TKLs decreased dramatically with expression of DEBS1 in both pikAIV and pilMIII-pikAIV deletion hosts (0.5 mg/L), but not DEBS1-TE, and could be partially restored by addition of a PikAIV complementation plasmid. These data suggest that PikAIV is able to efficiently catalyze formation of 6-membered lactone ring products from acyl-bound intermediates on DEBS1 in a manner analogous to that observed for 12-membered macrolide products from PikAIII. Significant sequence similarity and length of the C-terminal linker region of PikAIII and DEBS1 suggest that this region may be responsible for the interaction with PiRAIV. A replacement of this linker region of DEBS1 with the corresponding region of PikAI led to a 95% decrease in TKL levels in S. venezuelae, consistent with this hypothesis.