Institute of Molecular and Cell Biology, 61 Biopolis drive, Singapore, 138673, Singapore.
European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042, Grenoble, France.
Sci Rep. 2022 Mar 30;12(1):5353. doi: 10.1038/s41598-022-09188-8.
Non-ribosomal peptide synthetases (NRPS) are multi-modular/domain enzymes that catalyze the synthesis of bioactive peptides. A crucial step in the process is peptide elongation accomplished by the condensation (C) domain with the aid of a peptidyl carrier or thiolation (T) domain. Here, we examined condensation reaction carried out by NRPS AmbB involved in biosynthesis of L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) in P. aeruginosa. We determined crystal structures of the truncated T-C bidomain of AmbB in three forms, the apo enzyme with disordered T domain, the holo form with serine linked phosphopantetheine (Ppant) and a holo form with substrate (L-alanine) loaded onto Ppant. The two holo forms feature the T domain in a substrate-donation conformation. Mutagenesis combined with functional assays identified residues essential for the attachment of Ppant, anchoring the Ppant-L-Ala in the donor catalytic channel and the role of the conserved His953 in condensation activity. Altogether, these results provide structural insights into the condensation reaction at the donor site with a substrate-bound C domain of AmbB and lay the foundation for understanding the molecular mechanism of condensation which is crucial for AMB synthesis.
非核糖体肽合成酶（NRPS）是多模块/域酶，催化生物活性肽的合成。该过程的一个关键步骤是通过缩合（C）域在肽酰载体或硫醇化（T）域的辅助下完成肽的延伸。在这里，我们研究了参与铜绿假单胞菌中 L-2-氨基-4-甲氧基-trans-3-丁烯酸（AMB）生物合成的 NRPS AmbB 进行的缩合反应。我们确定了 AmbB 的截断 T-C 双域的三种形式的晶体结构，无规构象的 T 域的无酶形式、与丝氨酸相连的磷酸泛酰巯基乙胺（Ppant）的全酶形式以及底物（L-丙氨酸）加载到 Ppant 上的全酶形式。这两种全酶形式的 T 域均呈现出底物供体构象。突变体结合功能测定鉴定了与 Ppant 结合、锚定 Ppant-L-丙氨酸在供体催化通道中的附着以及保守的 His953 在缩合活性中的作用所必需的残基。总之，这些结果提供了结构上的见解，了解了与底物结合的 AmbB 的 C 域在供体部位的缩合反应，并为理解缩合的分子机制奠定了基础，这对于 AMB 的合成至关重要。
