Overexpression, purification and characterization of SimL, an amide synthetase involved in simocyclinone biosynthesis
Overexpression, purification and characterization of SimL, an amide synthetase involved in simocyclinone biosynthesis
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DOI：
10.1007/s00203-005-0770-0
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发表时间：
2005-05-01
影响因子：
2.8
通讯作者：
Heide, L
中科院分区：
文献类型：
--
作者：
Luft, T;Li, SM;Heide, L
西莫司酮D8是由抗生素链霉菌Tu 6040产生的一种有效的细菌旋转酶抑制剂。它含有氨基香豆素部分，类似于新香豆素的部分，它通过酰胺键连接到结构复杂的酰基部分，由芳香族环素聚酮基核、脱氧糖橄榄糖和四烯二元酸组成。我们现在已经研究了SIML酶，它负责西莫环酮的酰胺键的形成。克隆了该基因，并在变铅青链霉菌T7中进行了表达，纯化后的蛋白接近均一，并对其进行了鉴定。60 kDa的蛋白质既能催化依赖于ATP的酰基成分的激活，又能将其转移到氨基香豆素环的氨基上，而不需要4‘-磷酸铁蛋白辅因子。除了其天然底物西莫环酮C4外，SIML还接受一系列肉桂酸和苯甲酸的衍生物以及其他几种结构非常不同的酸。这些发现使SIML有可能成为创造新的氨基香豆素抗生素的工具。
Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tu 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4'-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.