Exploration of the regulation and structure-function relationships of enzymes and proteins involved in sulfur amino acid metabolism and storage in bacterial and plant systems

蛋白质工程和其他纳米科学在提供新的环境友好型技术以应对社会面临的一些挑战方面具有非凡的潜力。我的实验室为这项研究所需的跨学科方法做了很好的准备。我们独特的专业知识融合了蛋白质生物化学、微生物学和植物科学。由此产生的知识转化机会(例如在生物化学和植物科学之间)和所采用的技术的多样性提供了强有力的培训环境。我的研究计划的主要重点是氨基酸代谢，我们关注的是蛋白质结构与功能的关系。这些主题的无处不在的性质为我们在不同系统中的研究提供了基础，正如本提案中描述的两个正在进行的项目所表明的那样：1.工程吡哆醛5‘-磷酸酶。蛋白质是一种大的生物分子，由称为氨基酸的单个构建块组成，这些氨基酸以复杂的三维模式折叠。这使得活细胞内数千种不同蛋白质中的每一种都具有独特和特定的功能。蛋白质的众多作用之一是催化化学反应。酶是一种生物大分子，通常是蛋白质，它们催化生化反应，在活细胞的生理条件下将起始的“底物”转化为特定的“产物”。为生物技术量身定做酶的经济前景刺激了对蛋白质工程研究的投资和兴趣。2010年全球工业酶的市场价值为33亿美元，预计这一价值将稳步增长。催化氨基酸转化的酶一般依赖于吡哆醛5‘-磷酸(PLP)。PLP辅因子的多功能性使得它可以催化一系列反应。我们将继续我们的研究，探索这些酶特有的复杂和微妙的结构-功能关系。我们的长期目标是应用这些知识来修改它们的特性，以满足工业过程的要求。2.改善植物的氨基酸平衡，改善其营养特性。鹰嘴豆等豆类谷物约占世界膳食蛋白质供应量的10%，每年为加拿大创造约27亿加元的出口收入。然而，这些种子缺乏必需的氨基酸蛋氨酸，其含量平均比推荐的人体营养推荐的3-5%低5倍。据报道，改变特定作物收获部分的氨基酸含量的尝试可以归类为“推”或“拉”策略。PUSH方法改变了缺乏氨基酸的生物合成途径中的代谢物的流量，从而增加了可溶性氨基酸库。互补拉动策略着眼于终点，试图增加种子贮藏蛋白质中目标氨基酸的含量。我们正在努力：(1)在目标植物物种的背景下，彻底了解这些生物合成酶的调节，以便更巧妙地增加可溶蛋氨酸库；(2)建立两阶段筛选，使天然种子储存蛋白的工程能够修改其氨基酸组成。这一战略是新颖的，并为科学学科之间的知识转化提供了一个范例。我们的研究将为提高谷物豆类的营养品质提供所需的知识基础。我们创新的双相筛也有潜力应用于设计其他作物的氨基酸图谱(例如增加谷物的赖氨酸含量)。

Protein engineering and other nanoscale sciences possess remarkable potential to provide new environmentally friendly technologies to address a number of the challenges facing society. My lab is well positioned for the transdisciplinary approach required for this research. Our distinct blend of expertise spans protein biochemistry, microbiology and plant science. The resulting opportunities for knowledge translation (e.g. between biochemistry and plant science) and the diversity of techniques employed provide a strong training environment. The primary focus of my research program is amino acid metabolism and we have focused on protein structure-function relationships. The ubiquitous nature of these themes provides the basis for our studies in diverse systems, as illustrated by the two ongoing projects described in this proposal: 1. Engineering pyridoxal 5’-phosphate enzymes. Proteins are large biological molecules, formed of individual building blocks called amino acids, which fold in a complex three-dimensional pattern. This enables the unique and specific function of each of the thousands of distinct proteins inside a living cell. One of the many roles of proteins is in the catalysis of chemical reactions. Enzymes are large biological molecules, generally proteins, which catalyze biochemical reactions, converting the starting `substrate` to a specific `product` under the physiological conditions of the living cell. The economic prospects of tailoring enzymes for biotechnology have spurred investments and interest in protein engineering research. The 2010 global market value of industrial enzymes was $3.3 US billion, a value predicted to grow steadily. Enzymes catalyzing transformations of amino acids are generally dependent on pyridoxal 5’-phosphate (PLP). The versatility of the PLP cofactor is such that it can catalyze an array of reactions. We will continue our research probing the complex and subtle structure-function relationships that underlie specificity in these enzymes. Our long-term goal is the application of this knowledge to modify their specificity to meet the requirements of industrial processes. 2. Modifying the amino acid balance of plants to improve their nutritional properties. Grain legumes, such as chickpea, provide approximately 10% of the world supply of dietary protein and produce approximately $2.7 billion (CDN) in export revenue for Canada, annually. However, the seeds are deficient in the essential amino acid methionine, containing on average 5-fold less than the recommended 3-5% recommended for human nutrition. Reported attempts to modify the amino acid content of the harvested portion of a given crop plant can be classified as either ‘push’ or ‘pull’ strategies. Push approaches alter the flux of metabolites in the biosynthetic pathway of the deficient amino acid, thereby yielding an increase in the soluble amino acid pool. The complementary pull strategy focuses on the endpoint, attempting to increase the content of the target amino acid in the storage proteins of the seed. We are working to: (1) develop a thorough understanding of the regulation of these biosynthetic enzymes, in the context of the target plant species, in order to more subtly increase the soluble methionine pool and (2) establish a two-phase screen to enable engineering of the native seed storage proteins to modify their amino acid profile. This strategy is novel and provides an example of knowledge translation between scientific disciplines. Our research will provide the knowledge base required to improve the nutritional quality of grain legumes. Our innovative two-phase screen also has the potential to be applied to engineer the amino acid profiles of other crops (e.g. increasing the lysine content of cereals).