Structure/Activity Relationship of Pectate Lyase

果胶酸裂解酶的构效关系

• 批准号: 9408999
• 负责人: Noel Keen
• 金额: $ 30.4万
• 依托单位: University of California-Riverside
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9408999&HistoricalAwards=false
• 关键词: Structure; Activity; Relationship; Pectate; Lyase

Keen 9408999 The three-dimensional structures of two pectate lyase (Pel) proteins have been determined. These proteins are highly stable and are extracellularly secreted by the plant pathogenic bacterium, Erwinia chrysanthemi, to function as key virulence factors. The Pel structures are revolutionary, best described as a 'corkscrew' structure of parallel b strands, not previously observed or even predicted. The parallel b-strands are arranged in a helical structure with ca.22 amino acids per helical turn. The function s of the proteins reside on loops which protrude from the central helix. Since computer algorithms suggest that the parallel b-helix structure may be much more widespread in biology, further characterization of the pectate lyases is lucrative. Our immediate objectives are to characterize the calcium and substrate binding sites of the pectate lyases as well as to determine amino acid residues important in their folding and secretion. Considerable preliminary work has been directed to the development of over- expression systems for pel genes in E.coli and methods for efficiently obtaining oligonucleotide site directed mutations altering one or several specific amino acids. With structural refinement nearing completion for PelC and PelE as well as the ongoing development of fluorescence assays to monitor calcium and substrate binding, the investigators are in position to define mechanisms underlying catalysis, secretion and folding. The results will, in turn, open exciting future avenues. These include critical study of the pectate lyases as pathogen virulence factors; detailed work on their biological secretion; and use of the parallel b-helix as a carrier scaffold for synthetic antigen and catalytic peptide domains that may have utility in clinical medicine. %%% Pectate lyases are important pathogenicity factors released by several pathogens of plants. These enzymatically active proteins attack the integrity of the plant cell wall and per mit pathogen spread in plant tissues and result in damage. As such, these enzymes are a major contributor to the large world-wide loss of food and fiber to disease organisms. Recently, it was shown that a pectate lyases from the pathogenic bacterium, Erwinia chrysanthemi, has a novel structure at the atomic level. This structure, called the parallel beta-helix, is unlike any previously observed structural motif and has important implications on protein structure. The parallel beta-helix structure also has important ramifications for the biological function of pectate lyase in the degradation of plant cells. The present proposal is to study several aspects of the biology of pectate lyase by changing certain amino acids. These changes will be accomplished by techniques involving recombinant DNA technology. This will permit assessing the impact of the changes on the biological properties of the pectate lyase and should eventually allow understanding of the precise interaction of the enzyme with plant tissues. ***

Ken 9408999测定了两种果胶酸裂解酶(PEL)蛋白的三维结构。这些蛋白质高度稳定，由植物病原菌欧文氏菊花杆菌胞外分泌，发挥关键毒力因子的作用。PEL结构是革命性的，最好的描述是一种平行b链的开瓶器结构，以前没有观察到，甚至没有预测到。平行的b-链以螺旋结构排列，每个螺旋旋转约有22个氨基酸。这些蛋白质的功能S存在于从中央螺旋突出的环上。由于计算机算法表明，平行的b-螺旋结构在生物学中可能更加广泛，进一步表征果胶酸裂解酶是有利可图的。我们的近期目标是确定果胶裂解酶的钙和底物结合部位，以及确定在其折叠和分泌过程中重要的氨基酸残基。大量的前期工作已经被用于在E.Coli中开发Pel基因的过度表达系统和有效地获得改变一种或几种特定氨基酸的寡核苷酸定点突变的方法。随着PELC和PELE的结构优化接近完成，以及监测钙和底物结合的荧光分析技术的不断发展，研究人员已经能够确定催化、分泌和折叠的潜在机制。这一结果将反过来开辟令人兴奋的未来道路。这些措施包括作为病原体毒力因子的果胶裂解酶的关键研究；对其生物分泌的详细研究；以及使用平行的b-螺旋作为合成抗原和催化多肽域的载体支架，这些可能在临床医学中有实用价值。果胶裂解酶是多种植物病原菌释放的重要致病因子。这些具有酶活性的蛋白攻击植物细胞壁的完整性，并导致病原菌在植物组织中传播并造成损害。因此，这些酶是全球范围内疾病生物体大量损失食物和纤维的主要原因。最近的研究表明，从病原菌欧文氏杆菌中分离出来的果胶裂解酶在原子水平上具有新的结构。这种结构被称为平行的β-螺旋，不同于以前观察到的任何结构基序，对蛋白质结构具有重要影响。平行的β-螺旋结构对果胶裂解酶在植物细胞降解中的生物学功能也有重要的影响。目前的建议是通过改变某些氨基酸来研究果胶裂解酶生物学的几个方面。这些变化将通过涉及重组DNA技术的技术来实现。这将允许评估变化对果胶裂解酶生物学特性的影响，并最终应该允许了解酶与植物组织的精确相互作用。***