Genetic Variation of Adenylation Domains of the Anabaenopeptin Synthesis Operon and Evolution of Substrate Promiscuity
Genetic Variation of Adenylation Domains of the Anabaenopeptin Synthesis Operon and Evolution of Substrate Promiscuity
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DOI：
10.1128/jb.00360-11
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发表时间：
2011-08-01
影响因子：
3.2
通讯作者：
Kurmayer, Rainer
中科院分区：
文献类型：
--
作者：
Christiansen, Guntram;Philmus, Benjamin;Kurmayer, Rainer
anabaenopeptin （AP）是一种在蓝藻中以非核糖体合成的具有生物活性的环六肽。ap具有几个保守的基序，包括ureido键、位置5的n -甲基化和位置2的D-Lys。AP分子的所有其他位置都是可变的，导致许多结构变异。我们从agardhii浮游thrix CYA126/8菌株中鉴定了一个非核糖体肽合成酶（NRPS）操纵子，该操纵子由5个基因（apnA至apnE）编码6个NRPS模块，并通过apnC的插入失活产生突变体，证实了其在AP合成中的作用。为了将腺苷酸化结构域（A结构域）的遗传多样性与AP结构变异联系起来，我们对7株不同AP同系物产生的浮游thrix菌株的所有6个NRPS模块的A结构域进行了测序。值得注意的是，单个菌株共同产生含有化学上不同的氨基酸Arg和Tyr的ap，在外环位置1上。由于起始模块的A结构域（ApnA A1结构域）已被提出激活纳入外环位置1的氨基酸，我们决定从生化和系统发育两方面分析该结构域。在体外实验中发现，只有产生位置1含有Arg或Tyr的AP分子的菌株的ApnA A1酶才能激活这两种化学上不同的氨基酸。对ApnA A1结构域序列的系统发育分析表明，具有混杂ApnA A1结构域的菌株来源于仅激活精氨酸的祖先。令人惊讶的是，正选择似乎只影响apnA A1基因中的三个密码子，这表明这种显著的乱交只从点突变进化而来。
Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and D-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only.