Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis
Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis
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DOI：
10.1111/j.1432-1033.2004.04063.x
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发表时间：
2004-04-01
期刊：
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：
Linne, U;Schwarzer, D;Marahiel, MA
最近对参与微生物次级代谢的II型硫酯酶（TEII）的研究描述了这些酶在从聚酮酶和非核糖体肽合成酶的错误引发的全-（酰基载体蛋白）和全-（肽基载体蛋白）去除短酰基-S-磷酸泛酰巯基乙胺中间体中的作用。由于这类酶的结构信息的情况下，我们进行了突变分析的原型TEII必需的有效生产的脂肽抗生素表面活性素（TEIIisrf），这导致了催化和结构残基的鉴定。在16个TEIIs序列比对的基础上，构建了TEIIisrf高度保守残基的10个单突变体和1个双突变体，并进行了生化研究。我们清楚地确定了由Ser 86，Asp 190和His 216组成的催化三联体，表明TEIIsrf属于α/β-水解酶超家族。这些残基与脂肪族侧链的残基交换废除酶活性，而半胱氨酸的活性位点Ser 86的替换产生的酶的活性略有降低。与此相反，交换的第二个严格保守的天冬酰胺（Asp 163）与丙氨酸导致在一个活跃的，但不稳定的酶，排除了这个残基在催化作用，并建议结构功能。结果定义三个催化和至少一个结构残基在非核糖体肽合成酶TEII。
Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipopeptide antibiotic surfactin (TEIIsrf), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEIIsrf were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEIIsrf belongs to the alpha/beta-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a nonribosomal peptide synthetase TEII.