Characterization and Engineering of Fused Chorismate-Utilizing Enzymes

融合分支酸利用酶的表征和工程

• 批准号: 7585730
• 负责人: WALTON MALCOLM BYRNES
• 金额: $ 11.1万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7585730
• 关键词: 4-Aminobenzoic Acid; Achievement; Anabolism; Anthranilate Synthase; Antibiotics; Antifungal Antibiotics; Aromatic Amino Acids; Bacteria; Bioinformatics; Chloramphenicol; Engineering; Enzymes; Escherichia coli; Folate; Goals; Gram-Positive Bacteria; Metabolic; Modeling; Names; Pathway interactions; Phenazines; Plague; Reaction; Recombinants; Sequence Alignment; Site-Directed Mutagenesis; Streptomyces; Streptomyces griseus; Streptomycin; Structure; Tryptophan; Ubiquinone; Work; aminodeoxychorismate lyase; aminodeoxychorismate synthase; anthranilate; expectation; expression cloning; shikimate; tool

DESCRIPTION (provided by applicant): In bacteria, the shikimate pathway-derived intermediate chorismate is an important branch point for the synthesis of a number of primary and secondary metabolites. Examples include the aromatic amino acids, folate, coenzymes Q and the antibiotics chloramphenicol and candicidin, to name a few. There are five known chorismate-utilizing enzymes that catalyze reactions in pathways that branch off from chorismate. Two of these are anthranilate synthase with its associated aminodeoxyisochorismate (ADIC) synthase activity, and p-aminobenzoic acid (PABA) synthase with its associated aminodeoxychorismate (ADC) activity. Most anthranilate syntheses involved in tryptophan biosynthesis have distinct TrpE and TrpG subunits; ADC synthase contains PabA and PabB subunits. The work proposed here is directed toward the characterization or engineering of fused chorismate-utilizing enzymes. One such enzyme is from Streptomyces venezuelae (Sv), a chloramphenicol-producing Gram positive bacterium. A second is from Streptomyces griseus, producer of the antifungal antibiotic candicidin (and streptomycin). A third originates in Escherichia coli. There are three major aims or goals: (1) the detailed characterization of the structure and function of the fused SvTrpEG, which, contrary to initial expectations, has been found to be an anthranilate synthase as opposed to an ADIC synthase; (2) the cloning, expression and preliminary characterization of PabAB from S. griseus; and (3) the engineering of a fused E. coli PabAB enzyme. Achievement of the first goal will provide a deeper understanding of what structurally and functionally sets TrpEG apart from the homologous phenazine biosynthetic enzyme PhzE. Achievement of goal 2 will further an understanding of how PabA and PabB subunits of the metabolically important enzyme PABA synthase associate and work together to produce ADC, from which PABA is formed through the catalytic action of a third enzyme, ADC lyase or PabC. It could also aid in the metabolic engineering of the candicidin biosynthetic pathway in order to increase yield of antibiotic. Finally, achievement of the third goal may solve a problem that has plagued the study of the interaction between the PabA and PabB subunits of E. coli PABA synthase: their weak interaction when separate.

描述（由申请人提供）：在细菌中，透明途径衍生的中间脉络化是合成许多原代和次级代谢物的重要分支点。例子包括芳香氨基酸，叶酸，辅酶Q以及抗生素氯霉素和糖果素，仅举几例。有五种已知的假材利用酶，可催化从绒毛层分支的途径中的反应。其中两个是与其相关的氨基氧化异构酸盐（ADIC）合酶活性和p-氨基苯甲酸（PABA）合成酶及其相关的氨基氧化剂（ADC）活性的P-氨基苯甲酸（PABA）合成酶。色氨酸生物合成涉及的大多数炭疽菌合成具有独特的TRPE和TRPG亚基。 ADC合酶包含PABA和PABB亚基。此处提出的工作是针对融合融化酶的酶的表征或工程。一种这样的酶来自委内瑞斯链霉菌（SV），这是一种产生氯霉素的革兰糖阳性细菌。第二个来自抗真菌抗生素糖苷（和链霉菌素）的生产国链霉菌。三分之一起源于大肠杆菌。有三个主要的目的或目标：（1）融合SVTRPEG的结构和功能的详细表征，与初始期望相反，已被发现是炭疽酸合酶，而不是ADIC合酶； （2）从S. griseus的Pabab的克隆，表达和初步表征； （3）融合大肠杆菌pabab酶的工程。实现第一个目标将为您更深入地了解哪些结构和功能设置TRPEG与同源苯嗪生物合成酶PHZE不同。目标2的实现将进一步了解代谢上重要的酶PABA合酶的PABA和PABB亚基，并共同生产ADC，从中通过第三酶，ADC裂解酶或PABC的催化作用形成PABA。它还可以帮助糖果生物合成途径的代谢工程，以提高抗生素的产量。最后，第三个目标的实现可能解决了一个问题，困扰着大肠杆菌PABA合酶的PABA和PABB亚基之间的相互作用：分开时它们的弱相互作用。