Assembly line biosynthesis of bacterial siderophores

描述(申请人提供)：本研究的目的是了解细菌中肽类铁载体的生物合成机制。我们的范例是由鳗弧菌产生的两个多肽铁载体。一种是angibactin，它是质粒介导的铁摄取系统的重要组成部分，对这种细菌的毒力至关重要。另一种是Vanchrobactin，一种染色体编码的铁载体，在缺乏pJM1编码的angibactin系统的情况下，它成为鳗弧菌毒力系统的重要组成部分。在这种具有生物学意义的病原体中，控制这些铁载体生物合成的具体机制将进一步阐明。为了剖析这些机制，我们将结合使用遗传学和生物化学方法。具体研究目标为：1.深入剖析肌动蛋白生物合成过程中的流水线酶学机制。我们计划通过使用体外方法来确定不同结构域在angibactin生产的每个步骤中的作用。具体来说，我们将评估：来自AngB的ARCP结构域，与AngM的缩合(C)和(PPC)，AngR的半胱氨酸腺化结构域在激活半胱氨酸中的作用；AngN的每个Cy结构域在半胱氨酸与2，3-二羟基苯甲酸(DHBA)缩合和环合生成二羟基苯基噻唑(DHPT)中的作用；通过AngM的C结构域将DHPT转移到N-羟基组胺。2.NRPS之间是否通过特定的域进行通信？我们的证据表明，在pJM1质粒上和鳗弧菌的染色体上都冗余地编码了几个angibactin生物合成基因，其中一个基因angA只是染色体介导的。我们最近的工作表明，冗余基因和ANGA是由染色体基因簇的生物合成装置共享的，该基因簇干预了Vanchrobactin(N-[N‘-(2，3-二羟基苯甲酰基)-精氨基]丝氨酸)的产生和对其铁络合物的摄取，包括VabE、VabB、VabD和VabF以及其他剪裁酶。质粒介导的AngB和染色体上的VabB都是NRPSs，每个都有两个结构域：一个同裂裂解酶和一个ARCP，它们作为DHBA激活分子的第一受体，在angibactin(AngB)和Vanchrobactin(VabB)的生物合成中发挥作用。由于氨基和羧基末端的延伸，VabB比AngB稍大。虽然VabB在Vanchrobactin生物合成中起作用，但不能取代AngB在angibactin生物合成中的作用。我们认为这些结果是由于VabB和AngB中存在特定的通信介导域，使它们只能与其特定的NRPS相互作用，VabB的VabF和AngB的AngM。我们建议进行实验，以了解这些NRPS之间的蛋白质-蛋白质通讯的基础，从而促进多酶复合体中的选择性相互作用。我们的结果可能导致在铁载体生物合成过程中NRPSs通信这一迷人领域的新途径的探索。这些新的方法可能会导致更好地理解细菌中铁载体的生物合成和毒力。与公共卫生相关：我们打算将病原菌的非核糖体多肽合成酶系统剖析为一组特征良好的、可互换的模块结构域，这些结构域可用作设计的生物合成的催化试剂，这些药物和化疗产品可用于对抗由病原体引起的疾病。这些方法可能会导致在细菌的铁载体生物合成和毒力过程中探索新的途径。

DESCRIPTION (provided by applicant): The goal of this research is to understand the mechanism of biosynthesis of peptide siderophores in bacteria. Our paradigms are two peptide siderophores produced by the pathogen Vibrio anguillarum. One is anguibactin, an important component of a plasmid-mediated iron uptake system that is essential for virulence of this bacterium. The other is vanchrobactin, a chromosomally-encoded siderophore that in the absence of the pJM1-encoded anguibactin system becomes an important component of the virulence repertoire of V. anguillarum. The specific mechanisms that govern the biosynthesis of these siderophores in this biologically significant pathogen will be further elucidated. In order to dissect these mechanisms we will use a combination of genetic and biochemical approaches. The specific aims are: 1. Dissection of the mechanisms of assembly line enzymology in anguibactin biosynthesis. We plan to ascertain by using in vitro approaches the role of the different domains in each of anguibactin production steps. Specifically, we will assess: the role of the ArCP domain from AngB, the condensation (C) and (PPC) with AngM, the cysteine adenylation domain of AngR in activating cysteine; each Cy domain of AngN in cysteine condensation with 2,3-dihydroxybenzoic acid (DHBA) and cyclization to generate dihydroxyphenylthiazolyn (DHPT); the transfer of DHPT to N-hydroxy- histamine by the C domain of AngM. 2. Do NRPSs communicate with each other through specific domains? Our evidence indicates that several anguibactin biosynthetic genes are encoded redundantly on both the pJM1 plasmid and the chromosome of V. anguillarum and that one, angA is only chromosomally mediated. Our recent work demonstrated that the redundant genes and angA are shared by the biosynthetic apparatus for a chromosomal gene cluster that intervenes in the production of vanchrobactin (N-[N'-(2,3- dihydroxybenzoyl)-arginyl]-serine) and uptake of its ferric complexes and that consists of VabE, VabB, VabD and VabF, as well as other tailoring enzymes. The plasmid-mediated AngB and the chromosomal VabB are NRPSs with two domains each: an isochorysmate lyase and an ArCP that work as the first acceptors of an activated molecule of DHBA in the biosynthesis of anguibactin (AngB) and vanchrobactin (VabB). VabB is slightly larger than AngB due to extensions at the amino and carboxy terminus. Although VabB operates in vanchrobactin biosynthesis it cannot replace AngB in anguibactin biosynthesis synthesis. We believe that these results are due to the presence of specific communication-mediating domains in VabB and AngB that allow them to only interact with their specific NRPSs, VabF for VabB and AngM for AngB. We propose experiments to understand the basis of protein-protein communication between these NRPSs that facilitates the selective interaction in the multienzyme complexes. Our results could lead to the exploration of new avenues in the fascinating field of NRPSs communication during siderophore biosynthesis. These novel approaches will likely lead to a better understanding of siderophore biosynthesis and virulence in bacteria. PUBLIC HEALTH RELEVANCE: We intend to dissect the nonribosomal peptide synthetase systems of pathogenic bacteria into sets of well characterized, interchangeable modular domains that can be used as catalytic reagents for the designed biosynthesis of novel pharmacological and chemotherapeutic products that can be used to combat diseases caused by pathogenic bacteria. These approaches will likely lead to the exploration of new avenues in the process of siderophore biosynthesis and virulence in bacteria.