Miyazawa Takeshi, Takahashi Shunji, Kawata Akihiro, Panthee Suresh, Hayashi Teruo, Shimizu Takeshi, Nogawa Toshihiko, Osada Hiroyuki
RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and; the Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and.
J Biol Chem. 2015 Nov 6;290(45):26994-27011. doi: 10.1074/jbc.M115.677195. Epub 2015 Sep 16.
Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8-C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via β-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the β-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-(13)C]octanoic acid. (13)C NMR analysis clearly demonstrated incorporation of the [3,4-(13)C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.
了解非典型聚酮延伸单元的生物合成机制对于生物活性天然产物的开发至关重要。链霉菌属SN-593产生的瑞弗霉素（RM）衍生物具有多个脂肪族延伸单元。在此，我们通过分析与巴豆酰辅酶A羧化酶/还原酶同源物revT基因形成转录单元的revR和revS基因，研究了2-烷基丙二酰辅酶A形成的分子基础。我们主要关注未表征的腺苷酸形成酶（RevS）。revS基因破坏导致所有RM衍生物减少，而该基因的重新引入恢复了RM的产量。尽管基于系统发育分析RevS被归类于脂肪酰-AMP连接酶进化枝，但生化特性表明该酶催化中链脂肪酰辅酶A连接酶（FACL）活性而非脂肪酰-AMP连接酶活性，这表明了酰基辅酶A生物合成的分子进化。此外，我们使用纯化的RevS和RevT检测了脂肪酸体外转化为2-烷基丙二酰辅酶A的过程。偶联反应显示己烯酸高效转化为丁基丙二酰辅酶A。RevS有效催化C8 - C10中链FACL活性；因此，我们推测酰基辅酶A前体通过β-氧化被截短并转化为RevT底物（E）-2-烯酰辅酶A。为了确定β-氧化过程是否参与RevS和RevT反应之间，我们使用[1,2,3,4-（13）C]辛酸进行了饲喂实验。（13）C NMR分析清楚地证明了[3,4-（13）C]辛酸部分掺入了RM-A的结构中。我们的结果为未表征的RevS同源物的作用提供了见解，这些同源物可能催化中链FACL以产生独特的聚酮延伸单元。
