A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth
A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth
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DOI：
10.1073/pnas.0806305105
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发表时间：
2008-09-02
影响因子：
11.1
通讯作者：
Magarvey, Nathan A.
中科院分区：
文献类型：
--
作者：
Calderone, Christopher T.;Bumpus, Stefanie B.;Magarvey, Nathan A.
在非核糖体肽合成酶（NRPS）和聚酮合成酶（IRKS）模块与催化结构域一起作用于反式装配线上，聚酮信号代谢产物bacillaene和dihydrobacillaene在枯草芽孢杆菌的酶装配线上进行生物合成。这些信号代谢产物具有不寻常的起始单位α -羟基异己酸酯（α - hic）。我们在这里表明，它是由PksJ（一种杂交PKS/NRPS）的第一个腺苷化结构域初始激活α -酮异己酸酯（α - kic）并安装在邻近的硫基化(T)结构域的pantetheinyl上引起的。质谱分析表明，a-KIC单元被PksJ中的第二个NRPS模块拉长为α - kic - gly。PksJ的第三个模块使用PKS逻辑，包含一个嵌入式酮还原酶（KR）结构域以及两个相邻的T结构域。我们发现这个KR结构域减少了典型的3-酮丁基链，也减少了PksJ t结构域双偶体上含有α - kic的中间体的α -酮基。这种KR活性解释了在二氢硅酸菌烯/硅酸菌烯对中发现的α - hic部分，并代表了装配线双功能α -和β -KR作用于生长链中间体的不同位置的例子。
The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (IRKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit alpha-hydroxyisocaproate (alpha-HIC). We show here that it arises from initial activation of alpha-ketoisocaproate (alpha-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The a-KIC unit is elongated to alpha-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the alpha-keto group of alpha-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the alpha-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function alpha- and beta-KR acting on disparate positions of a growing chain intermediate.