Dipeptide synthesis by internal adenylation domains of a multidomain enzyme involved in nonribosomal peptide synthesis
Dipeptide synthesis by internal adenylation domains of a multidomain enzyme involved in nonribosomal peptide synthesis
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DOI：
10.2323/jgam.2018.03.001
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发表时间：
2019-01-01
影响因子：
1.2
通讯作者：
Kobayashi, Michihiko
中科院分区：
文献类型：
--
作者：
Abe, Tomoko;Kobayashi, Kenta;Kobayashi, Michihiko
非核糖体肽合成酶（NRPS）的腺苷酸化结构域负责其在ATP消耗时的选择性底物识别和底物活化（产生酰基-O-AMP中间体）。DhbF是一种参与芽孢杆菌素合成的NRPS，由多个结构域[腺苷酸化结构域、缩合结构域、肽基载体蛋白（PCP）结构域和硫酯酶结构域：DhbFA 1和DhbFA 2（此处命名）是多结构域酶DhbF中的“内部”腺苷酸化结构域。我们首次成功地分别表达和纯化了“内部”腺苷酸化结构域DhbFA 1和DhbFA 2。此外，我们最初证明了二肽合成的“内部”腺苷酸化结构域。当甘氨酸和L-半胱氨酸被用作DhbFA 1的底物时，观察到N-甘氨酰L-半胱氨酸（Gly-Cys）的形成。此外，当L-苏氨酸和L-半胱氨酸被用作DhbFA 2的底物时，形成N-L-苏氨酰-L-半胱氨酸（Thr-Cys）。这些发现表明，两个腺苷酸化结构域通过形成包含氨基酸的羧基和L-半胱氨酸的氨基的碳-氮键来产生二肽，尽管这些腺苷酸化结构域是使用4 '-磷酸泛酰巯基乙胺（结合到PCP结构域）作为底物的酸-硫醇连接酶。此外，DhbFA 1和DhbFA 2合成寡肽以及二肽。
The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for its selective substrate recognition and activation of the substrate (yielding an acyl-O-AMP intermediate) on ATP consumption. DhbF is an NRPS involved in bacillibactin synthesis and consists of multiple domains [adenylation domain, condensation domain, peptidyl carrier protein (PCP) domain, and thioesterase domain: DhbFA1 and DhbFA2 (here named) are "internal" adenylation domains in the multidomain enzyme DhbF. We firstly succeeded in expressing and purifying the "internal" adenylation domains DhbFA1 and DhbFA2 separately. Furthermore, we initially demonstrated dipeptide synthesis by "internal" adenylation domains. When glycine and L-cysteine were used as substrates of DhbFA1, the formation of N-glycylL-cysteine (Gly-Cys) was observed. Furthermore, when L-threonine and L-cysteine were used as substrates of DhbFA2, N-L-threonyl-L-cysteine (Thr-Cys) was formed. These findings showed that both adenylation domains produced dipeptides by forming a carbon-nitrogen bond comprising the carboxyl group of an amino acid and the amino group of L-cysteine, although these adenylation domains are acid-thiol ligase using 4'-phosphopantetheine (bound to the PCP domain) as a substrate. Furthermore, DhbFA1 and DhbFA2 synthesized oligopeptides as well as dipeptides.