Biochemical characterization of peptides carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases
Biochemical characterization of peptides carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases
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DOI：
10.1016/s1074-5521(96)90180-5
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发表时间：
1996-11-01
影响因子：
--
通讯作者：
Marahiel, MA
中科院分区：
文献类型：
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作者：
Stachelhaus, T;Huser, A;Marahiel, MA
背景资料：一组结构多样的生物活性肽是由肽合成酶合成的，肽合成酶作为生长肽链的模板，通过硫酯键连接到酶上。蛋白质模板由独特的底物激活模块组成，其顺序决定了相应肽产物的一级结构。每个模块包含催化腺苷酸化、硫酯和肽键形成以及底物修饰的限定结构域。为了证明一个假定的巯基化结构域（PCP）参与非核糖体肽合成过程中氨基酰基残基的共价结合和转移，我们克隆了酪氨酸合成酶1（TycA）的一个区域，并对其进行了生化表征。结果：利用其与脂肪酸和聚酮生物合成的酰基载体蛋白基因的同源性，克隆了编码PCP的327 bp基因片段。该蛋白表达为His（6）融合蛋白，并通过亲和层析在一个步骤中纯化。β-[H-3]丙氨酸（辅酶A的前体）的掺入证明了PCP被辅因子4 '-磷酸泛酰巯基乙胺修饰。当存在一个腺苷酸化结构域，以提供氨基腺苷酸moiety，PCP可以酰化在vitro.Conclusions：PCP可以共价结合到辅因子磷酸泛酰巯基乙胺，随后可以被酰化，强烈支持非核糖体肽合成的多载体模型。腺苷酸化和巯基化结构域可以各自充当独立的多功能酶，进一步证实了肽解酶的模块结构，并且还可以反式执行顺序步骤，如多酶复合物一样。
Background: A structurally diverse group of bioactive peptides is synthesized by peptide synthetases which act as templates for a growing peptide chain, attached to the enzyme via a thioester bond. The protein templates are composed of distinctive substrate-activating modules, whose order dictates the primary structure of the corresponding peptide product. Each module contains defined domains that catalyze adenylation, thioester and peptide bond formation, as well as substrate modifications. To show that a putative thiolation domain (PCP) is involved in covalent binding and transfer of amino acyl residues during non-ribosomal peptide synthesis, we have cloned and biochemically characterized that region of tyrocidine synthetase 1, TycA.Results: The 327-bp gene fragment encoding PCP was cloned using its homology to the genes for the acyl carrier proteins of fatty acid and polyketide biosynthesis. The protein was expressed as a His(6) fusion protein, and purified in a single step by affinity chromatography. Incorporation of beta-[H-3]alanine, a precursor of coenzyme A, demonstrated the modification of PCP with the cofactor 4'-phosphopantetheine. When an adenylation domain is present to supply the amino adenylate moiety, PCP can be acylated in vitro.Conclusions: PCP can bind covalently to the cofactor phosphopantetheine and can subsequently be acylated, strongly supporting the multiple carrier model of non-ribosomal peptide synthesis. The adenylation and thiolation domains can each act as independent multifunctional enzymes, further confirming the modular structure of peptide synthases, and can also perform sequential steps in trans, as do multienzyme complexes.