Protein Complexes in Alginate Maturation

海藻酸盐成熟中的蛋白质复合物

• 批准号: 8049062
• 负责人: Peter A Tipton
• 金额: $ 20.51万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-10 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049062
• 关键词: Acetyl Coenzyme A; Acetylation; Adherence; Affect; Alginates; Anabolism; Antibiotics; Antibodies; Bacteria; Biochemical; Carbohydrates; Cell membrane; Cells; Characteristics; Chemicals; Complement; Complex; Cytoplasm; Data; Energy-Generating Resources; Enzymes; Fluorescence Anisotropy; Gel Chromatography; Genetic; Genome; Gram-Negative Bacteria; Health; Host resistance; Human; Immune response; Immune system; Infection; Investigation; Kinetics; Lead; Learning; Life; Mediating; Membrane; Membrane Proteins; Metabolic; Methods; Microbial Biofilms; Modeling; Molecular; Multiprotein Complexes; Nature; Operon; Pathway interactions; Penetration; Phagocytosis; Phenotype; Play; Polymers; Polysaccharides; Prevention; Process; Production; Property; Proteins; Pseudomonas aeruginosa; Reaction; Reactive Oxygen Species; Role; Solubility; Solutions; Source; Staging; Structural Genes; Structure; Surface; System; Techniques; Time; Virulence; analytical ultracentrifugation; aqueous; capsule; data modeling; epimerase; epimerization; fascinate; insight; monomer; mutant; pathogen; pathogenic bacteria; patient population; periplasm; poly(beta-D-mannuronate) lyase; polymerization; porin; protein complex; research study; stoichiometry

DESCRIPTION (provided by applicant): Exopolysaccharides play important roles in determining the virulence of pathogenic bacteria, affecting their adherence to surfaces, and protecting them from antibiotics and the host's immune system. Much remains to be learned about the steps that govern the maturation and transport of exopolysaccharides in important human pathogens. The present application describes investigations into the final steps of alginate biosynthesis in Pseudomonas aeruginosa. Alginate is an unbranched heteropolymeric carbohydrate that forms a capsule around the bacteria. The precursors for polymer synthesis are generated in the cytoplasm, and polymerization to polymannuronan occurs through the action of integral cytoplasmic membrane proteins. We describe experiments to characterize the processes that occur between polymer formation and secretion. Polymannuronan undergoes epimerization at some residues via the action of a periplasmic enzyme. We have shown that another protein, AlgK, forms an isolable complex with the epimerase, and that complex formation requires a third protein AlgX. It is possible that the purpose of the complex is to protect the alginate from degradation by alginate lyase, and to facilitate the delivery of alginate to a porin protein on the outer membrane. During its time in the periplasm, alginate is partially acetylated. Three proteins have been shown to be required for acetylation, AlgI, AlgJ, and AlgF. Their roles remain undefined, and the acetylation process is enigmatic, since there is no acetyl-CoA present in the periplasm. We propose to characterize the protein complexes formed with the epimerase and to determine their influence on its functional properties, and to characterize the acetylation process in molecular detail. We will determine the source of the acetyl groups, and evaluate the roles of the separate proteins in the acetylation process. Kinetics techniques and biophysical methods for characterizing the protein complexes, including gel filtration chromatography, analytical ultracentrifugation and fluorescence anisotropy, will be employed. PUBLIC HEALTH RELEVANCE Pseudomonas aeruginosa is an opportunistic human pathogen that causes life-threatening infections in several different patient populations. A major contributing factor to the virulence of the bacteria and the persistence of their infections in the host is the exopolysaccharide alginate. Biochemical characterization of the steps that lead to formation of mature alginate may provide insights into how to block or ameliorate the effects of infection.

描述（由申请人提供）：外多糖在确定致病细菌的毒力，影响其对表面的粘附以及保护它们免受抗生素和宿主的免疫系统的影响中起着重要作用。关于在重要人类病原体中控制外多糖的成熟和运输的步骤还有很多尚待了解。本应用描述了对铜绿假单胞菌中藻酸盐生物合成的最后一步的研究。藻酸盐是一种未分支的异质源碳水化合物，在细菌周围形成胶囊。聚合物合成的前体是在细胞质中产生的，并且通过整合细胞质膜蛋白的作用发生聚合与聚熟曲唱。我们描述了表征聚合物形成和分泌之间发生的过程的实验。多曼努族人通过周质酶的作用在某些残基上进行表达。我们已经表明，另一种蛋白质ALGK形成了与分配酶的可分离复合物，并且该复合物形成需要第三个蛋白ALGX。该络合物的目的可能是保护藻酸盐免受藻酸裂解酶的降解，并促进藻酸盐在外膜上递送至孔蛋白蛋白。在周围的藻酸盐中，藻酸盐被部分乙酰化。已证明三种蛋白质是乙酰化，ALGI，ALGJ和ALGF所必需的。它们的作用保持不确定，并且乙酰化过程是神秘的，因为骨膜中没有乙酰辅酶A。我们建议表征用分配酶形成的蛋白质复合物，并确定它们对其功能特性的影响，并以分子细节表征乙酰化过程。我们将确定乙酰基的来源，并评估单独的蛋白在乙酰化过程中的作用。动力学技术和生物物理方法用于表征蛋白质复合物，包括凝胶过滤色谱，分析性超速离心和荧光各向异性。公共卫生相关性铜绿假单胞菌是一种机会主义的人类病原体，可在几个不同的患者人群中引起威胁生命的感染。导致细菌毒力及其在宿主中感染的持久性的主要因素是外多糖藻酸盐。导致成熟藻酸盐形成的步骤的生化表征可以提供有关如何阻止或改善感染影响的见解。

项目成果

Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.

AlgL（一种来自铜绿假单胞菌的藻酸盐裂解酶）的功能表征。

• DOI: 10.1021/bi301425r
• 发表时间: 2012-12-21
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Farrell EK;Tipton PA
• 通讯作者: Tipton PA

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX.

• DOI: 10.1107/s1744309110011851
• 发表时间: 2010-05-01
• 期刊: Acta crystallographica. Section F, Structural biology and crystallization communications
• 作者: Weadge JT;Yip PP;Robinson H;Arnett K;Tipton PA;Howell PL
• 通讯作者: Howell PL