BlmIII and BImIV nonribosornal peptide synthetase-catalyzed biosynthesis of the bleomycin bithiazole moiety involving both in Cis and in trans aminoacylation
BlmIII and BImIV nonribosornal peptide synthetase-catalyzed biosynthesis of the bleomycin bithiazole moiety involving both in Cis and in trans aminoacylation
复制标题
DOI：
10.1021/bi034817r
复制
发表时间：
2003-08-19
期刊：
影响因子：
2.9
通讯作者：
Shen, B
中科院分区：
文献类型：
--
作者：
Du, LC;Chen, M;Shen, B
博来霉素（BLM）生物合成基因簇的克隆和序列分析预测，两个非核糖体肽合成酶（NRPS），BlmIV和BlmIII，负责BLM双噻唑部分的生物合成。BlmIV是七结构域（C-2-A（2）-PCP 2-Cy-1-A（1）-PCP 1-Cy-0）NRPS，BlmIII是三结构域（A（0）-PCP 0-Ox）NRPS。位于BlmIV亚基上的Cy-1-A（1）-PCP 1的三个结构域、位于BlmIV亚基上的Cy-0的四个结构域和位于BlmIII亚基上的A（0）-PCP 0-Ox分别构成两个形成噻唑的NRPS-1和NRPS-0模块。预测BlmIII-A（0）是无功能的，这提出了NRPS-0模块如何激活Cys底物并将Cys底物加载到其同源BlmIII-PCP 0的问题。NRPS-0模块由位于两个不同亚基上的结构域组成，需要精确的蛋白质-蛋白质相互作用。在这里，我们报告的生产的BlmIV和BlmIII NRPS作为一个切除的域（S），模块，或完整的亚基形式和生化特性的所得酶在体外的BLM双噻唑生物合成中的作用。我们的结果（a）证实了BlmIII-A（0）是一种天然存在的无功能突变体，（B）证明了BlmIV-A（1）激活Cys并催化BlmIV-PCP 1的顺式氨酰化（对于NRPS-1）和BlmIII-PCP 0的反式氨酰化（对于NRPS-0），和（c）揭示了BlmIV亚基的C-末端，其特征在于前所未有的AGHDDD（G）和PGHDDG重复，是BlmIII-PCP 0的反式氨酰化绝对需要的。这些发现强调了NRPS在结构和天然产物生物合成机制方面的灵活性和多功能性，并为研究NRPS装配线酶学中的分子识别和蛋白质-蛋白质相互作用机制提供了一个极好的机会。
Cloning and sequence analysis of the bleomycin (BLM) biosynthetic gene cluster predicted that the two nonribosomal peptide synthetases (NRPSs), BlmIV and BlmIII, are responsible for the biosynthesis of the BLM bithiazole moiety. BlmIV is a seven domain (C-2-A(2)-PCP2-Cy-1-A(1)-PCP1-Cy-0) NRPS, and BlmIII is a three domain (A(0)-PCP0-Ox) NRPS. The three domains of Cy-1-A(1)-PCP1 residing on the BlmIV subunit, the four domains of Cy-0 residing on the BlmIV subunit, and A(0)-PCP0-Ox residing on the BlmIII subunit constitute the two thiazole-forming NRPS-1 and NRPS-0 modules, respectively. BlmIII-A(0) was predicted to be nonfunctional, raising the question of how the NRPS-0 module activates and loads the Cys substrate to its cognate BlmIII-PCP0. The NRPS-0 module consists of domains residing on two different subunits, requiring precise protein-protein interaction. Here, we report the production of the BlmIV and BlmIII NRPSs as an excised domain(s), module, or intact subunit form and biochemical characterizations of the resultant enzymes in vitro for their roles in BLM bithiazole biosynthesis. Our results (a) confirm that BlmIII-A(0) is a naturally occurring nonfunctional mutant, (b) demonstrate that BlmIV-A(1) activates Cys and catalyzes both in cis aminoacylation of BlmIV-PCP1 (for NRPS-1) and in trans aminoacylation of BlmIII-PCP0 (for NRPS-0), and (c) reveal that the C-terminus of the BlmIV subunit, characterized by the unprecedented AGHDDD(G) and PGHDDG repeats, is absolutely required for in trans aminoacylation of BlmIII-PCP0. These findings underscore the flexibility and versatility of NRPSs in both structure and mechanism for natural product biosynthesis and provide an outstanding opportunity to study the molecular recognition and protein-protein interaction mechanism in NRPS assembly line enzymology.