Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase.
Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase.
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DOI：
10.1074/jbc.m117.783464
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发表时间：
2017-06-30
期刊：
影响因子：
--
通讯作者：
Berteau O
中科院分区：
文献类型：
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作者：
Benjdia A;Decamps L;Guillot A;Kubiak X;Ruffié P;Sandström C;Berteau O
自由基S-腺苷甲硫氨酸（SAM）酶作为生物催化剂的主要超家族而出现，其参与生物活性肽（称为核糖体合成和后修饰肽（RIPPs））的大家族的生物合成。这些酶已被证明催化非常规反应，如甲基转移到亲电碳原子，硫转移到Cα原子硫醚键或碳-碳键形成。最近，一种新的自由基SAM酶催化形成的赖氨酸-色氨酸键已被确定在嗜热链球菌，并提出了反应机理。通过结合定点诱变，生化测定和光谱分析，我们在这里表明，这种酶，属于新兴的家庭SPASM域自由基SAM酶，可能包含三个[4Fe-4S]簇。值得注意的是，我们的数据支持，七个保守的半胱氨酸残基，存在于SPASM结构域，是酶活性的关键。此外，我们发现了最低底物要求，并证明KW环肽比预期的更广泛，特别是在病原菌中。最后，我们显示了一个严格的特异性的酶的赖氨酸和色氨酸残基和依赖的8个氨基酸的前导肽的活性。总而言之，我们的研究表明SPASM结构域自由基SAM酶之间存在新的机制联系，并支持非半胱氨酸配体参与辅助簇的协调。
Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.