Assembly line biosynthesis of bacterial siderophores

细菌铁载体的装配线生物合成

• 批准号: 7922904
• 负责人: JORGE H CROSA
• 金额: $ 9.74万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922904
• 关键词: Acids; Anabolism; Bacteria; Biochemical; Chimera organism; Chromosomes; Communication; Complex; Cyclization; Cysteine; Disease; Dissection; Enzymatic Biochemistry; Enzymes; Gene Cluster; Genes; Genetic; Goals; Histamine; In Vitro; Iron; Lead; Lyase; Mediating; Multienzyme Complexes; Peptide Biosynthesis; Peptides; Physical condensation; Physiological; Plasmids; Process; Production; Proteins; Reaction; Reagent; Research; Role; Serine; Siderophores; System; Vibrio; Vibrio cholerae; Virulence; Work; anguibactin; base; combat; comparative; design; fascinate; gene cloning; genetic analysis; novel; novel strategies; pathogen; pathogenic bacteria; peptide synthase; polypeptide; public health relevance; research study; thioester; uptake

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.

描述（由申请人提供）：本研究的目的是了解细菌中肽铁载体的生物合成机制。我们的范例是由病原体鳗弧菌产生的两种肽铁载体。一种是anguibactin，它是质粒介导的铁吸收系统的重要组成部分，对该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，在没有pJM 1编码的anguibactin系统的情况下，它成为鳗弧菌毒力库的重要组成部分。将进一步阐明这种生物学上重要的病原体中这些铁载体生物合成的具体机制。为了剖析这些机制，我们将使用遗传和生物化学方法的组合。具体目标是：1. anguibactin生物合成的装配线酶学机制剖析。我们计划通过使用体外方法来确定不同结构域在每个anguibactin生产步骤中的作用。具体而言，我们将评估：来自AngB的ArCP结构域的作用，与AngM的缩合（C）和（PPC），AngR的半胱氨酸腺苷酸化结构域在活化半胱氨酸中的作用; AngN的每个Cy结构域在与2，3-二羟基苯甲酸（DHBA）的半胱氨酸缩合和环化以产生二羟基苯基噻唑啉（DHPT）中的作用; DHPT通过AngM的C结构域转移到N-羟基-组胺。2. NRPS通过特定的域相互通信吗？我们的证据表明几个anguibactin生物合成基因在pJM 1质粒和鳗弧菌染色体上都是冗余编码的，其中一个是angA，它只由染色体介导。我们最近的工作表明，冗余基因和angA由染色体基因簇的生物合成装置共享，该染色体基因簇干预vanchrobactin（N-[N '-（2，3-二羟基苯甲酰基）-乙酰基]-丝氨酸）的产生和其铁络合物的摄取，并且由VabE、VabB、VabD和VabF以及其他剪裁酶组成。质粒介导的AngB和染色体VabB是具有两个结构域的NRPS：异氯酸裂解酶和ArCP，其在anguibactin（AngB）和vanchrobactin（VabB）的生物合成中作为DHBA的活化分子的第一受体工作。由于在氨基和羧基末端的延伸，VabB略大于AngB。虽然VabB在万古菌素生物合成中起作用，但它不能在anguibactin生物合成中取代AngB。我们认为这些结果是由于VabB和AngB中存在特定的通讯介导结构域，使它们只能与其特定的NRPS相互作用，VabB的VabF和AngB的AngM。我们提出的实验，以了解这些NRPS之间的蛋白质-蛋白质通信的基础上，促进多酶复合物中的选择性相互作用。我们的研究结果可能会导致探索新的途径，在迷人的领域NRPSs通信在铁载体生物合成。这些新的方法可能会导致更好地了解细菌中的铁载体生物合成和毒力。公共卫生关系：我们打算将病原菌的非核糖体肽合成酶系统解剖成一组特征良好的、可互换的模块化结构域，这些结构域可用作催化剂，用于设计新的药理学和化学治疗产品的生物合成，这些产品可用于对抗病原菌引起的疾病。这些方法可能会导致在细菌中的铁载体生物合成和毒力的过程中的新途径的探索。