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首页> 外文期刊>Biochemistry >Mechanistic Investigations of the Dehydration Reaction of Lacticin 481 Synthetase Using Site-Directed Mutagenesis
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Mechanistic Investigations of the Dehydration Reaction of Lacticin 481 Synthetase Using Site-Directed Mutagenesis

机译：使用定点诱变的乳酸素481合成酶脱水反应的机理研究

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摘要

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lysl59 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.
机译：羊毛硫抗生素合成酶分别催化其肽底物中的Ser和Thr残基脱水为脱氢丙氨酸（Dha）和脱氢丁氨酸（Dhb），然后将Cys残基共轭添加到Dha和Dhb残基中，从而生成硫醚交联羊毛硫氨酸和甲基羊毛硫氨酸。在这项研究中，十个保守残基在表征最充分的家族成员乳酸素481合成酶（LctM）的脱水酶结构域中发生了突变。 His244和Tyr408的突变不会影响LctA底物的脱水活性，而Asn247，Glu261和Glu446的突变会大大减缓脱水并导致转化不完全。 Lysl59的突变减慢了净脱水的两个步骤：Ser / Thr残基的磷酸化和随后的磷酸盐消除步骤，形成了脱氢氨基酸。 Arg399突变为Met或Leu导致具有磷酸化活性，但显示出大大降低的磷酸盐消除活性的突变体。但是，Arg399Lys突变体保留了两种活性。类似地，Thr405Ala突变体使LctA底物磷酸化，但消除活性受损。最后，Asp242或Asp259突变为Asn导致突变酶缺乏可检测的脱水活性。 Asp242Asn突变体保留了磷酸盐消除活性，而Asp259Asn突变体无法从磷酸化底物肽中消除磷酸盐。提出了解释这些突变酶的表型的模型。

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