Generation of Unsaturated Alpha-keto Acids using Engineered Acetoacetate Decarboxylase-Like Enzymes

智力优势。乙酰乙酸脱羧酶样超家族（ADCSF）是一类新的酶，可能是新型“绿色化学”生物催化剂的来源。该项目的长期目标是确定选定的ADCSF酶中负责反应和底物特异性的结构特征，这些酶具有在研究或工业环境中作为有用的生物催化剂的潜力。有了这些知识，最终有可能将这些酶设计用于特定应用。本工作的目的是确定ADCSF酶Sbi_00515从链霉菌bingchengensis和SWIT_4259从鞘氨醇单胞菌wittichii的催化和底物特异性的机制。我们的中心假设是，这两种酶都是水合酶-醛缩酶，使用涉及赖氨酸希夫碱的机制催化α-酮酸的形成或分解。这些实验的基本原理是，由于这两种酶不具有脱羧酶活性，乙酰乙酸脱羧酶的结构必须已被改造以执行其他催化功能。对决定反应特异性的结构方面的透彻理解不仅有助于将这些酶作为生物催化剂进行工程设计，而且还将拓宽我们对酶结构与功能之间关系的理解。这些目标将通过实现以下具体目标来实现。首先，将确定推定的水合酶-醛缩酶Sbi_00515的优选底物和催化机制。将筛选Sbi_00515对一组潜在底物的活性。这些动力学实验将得到酶与潜在底物结合的结构研究的支持。野生型和突变体形式的Sbi_00515的瞬态和稳态动力学，连同动力学同位素效应，将用于探测催化机制。其次，将定义由Swit_4259催化的反应，并且将鉴定有利于C-C键断裂或形成的结构特征。X射线晶体学、稳态动力学和生物信息学方法将用于表征Swit_4259与候选底物的相互作用。Swit_4259和Sbi_00515活性位点之间的显著差异的观察表明，这两种酶具有不同的底物特异性，并可能催化不同的反应。两种酶的未配体和底物结合形式的结构的比较应该使我们能够辨别这些酶的结构和功能之间的关系。这项工作是原创的，因为它将扩展ADCSF领域，超越迄今为止研究的少数脱羧酶。由于缺乏对这个超家族的研究，实际上不可能预测通过基因组挖掘发现的家族成员的功能。这项研究的结果将是关于Sbi_00515催化的羟醛缩合和不依赖于辅因子的脱水的详细机制信息，这将有助于我们了解进化如何修改脱羧酶支架以执行新的催化功能。还预期将鉴定底物特异性的主要决定因素，这将促进旨在工程化Sbi_00515和Swit_4259以产生具有定制侧链的α-酮酸的后续工作。这些成果将通过扩展和帮助定义新兴的ADCSF领域，以及提高研究人员更准确地预测通过基因组测序鉴定的ADCSF酶的功能的能力，产生积极的影响。该项目将采用若干战略来促进教学、培训和学习。首先，PI的团队在研究项目中包括本科生方面有着既定的记录。结构酶学的工作建议很容易分为学期的子项目，让本科生作出重大贡献的研究进展。PI还将把实际的研究数据纳入他每年教授的两门课程，这两门课程主要是本科课程。这些数据将用于说明数据解释的原则，以及蛋白质结构和功能的基本方面。将特别关注由本提案资助的研究生和博士后学者的全面发展，不仅作为研究人员，而且作为科学界的全面整合成员。为了实现这一目标，所有学员将接受书面和口头介绍研究成果，学术诚信和专业发展的指导。为了最大限度地提高从拟议研究中受益的学生人数，PI正在参加密尔沃基工程学院提供的“学生建模研究主题”（SMART）计划。智能团队介绍10 - 12年级学生结构生物学，因为他们与他们的老师和教师的研究导师合作。最后，PI将通过一系列播客向科学界和公众传播研究结果。因此，本研究将产生积极的社会影响和应用效益，推动ADCSF领域的发展。

Intellectual Merit. The acetoacetate decarboxylase-like superfamily (ADCSF) is a new and largely unexplored group of en-zymes that may be a source of new "green chemistry" biocatalysts. The long-term goal of this project is to define the structural features responsible for reaction and substrate specificity in selected ADCSF enzymes with potential as useful biocatalysts in research or industrial settings. With this knowledge, it may ultimately be possible to engineer these enzymes for specific applications. The objective of this work is to determine the mechanisms of catalysis and substrate specificity in the ADCSF enzymes Sbi_00515 from Streptomyces bingchenggensis and Swit_4259 from Sphingomonas wittichii. Our central hypothesis is that both enzymes are hydratase-aldolases, catalyzing the formation or breakdown of alpha-keto acids using a mechanism involving a lysine Schiff base. The rationale for these experiments is that, since these two enzymes do not have decarboxylase activity, the acetoacetate decarboxylase structure must have been adapted to perform other catalytic functions. A thorough understanding of the structural aspects that determine reaction specificity will not only facilitate efforts to engineer these enzymes as biocatalysts, but will also broaden our understanding of the relationship between enzyme structure and function. These objectives will be attained by pursuing the following specific aims. First, the preferred substrate and catalytic mechanism of the putative hydratase-aldolase Sbi_00515 will be determined. Sbi_00515 will be screened for activity against a panel of potential substrates. These kinetic experiments will be supported by structural studies of the enzyme with potential substrates bound. Transient and steady state kinetics on wild-type and mutant forms of Sbi_00515, together with kinetic isotope effects, will be used to probe the catalytic mechanism. Second, the reaction catalyzed by Swit_4259 will be defined, and structural features favoring C-C bond breaking or formation will be identified. X-Ray crystallography, steady-state kinetics, and bioinformatics approaches will be used to characterize the interaction of Swit_4259 with candidate substrates. The observation of significant differences between the Swit_4259 and Sbi_00515 active sites suggests that the two enzymes have different substrate specificities and may catalyze different reactions. Comparisons of the structures of unliganded and substrate-bound forms of both enzymes should allow us to discern relationships between structure and function in these enzymes. This work is original, because it will expand the ADCSF field beyond the few decarboxylases studied thus far. Due to the dearth of research on this superfamily, it is virtually impossible to predict the functions of family members discovered by genome mining. The outcomes of the research will be detailed mechanistic information about Sbi_00515-catalyzed aldol condensation and cofactor-independent dehydration, which will help us understand how evolution has modified the decarboxylase scaffold to perform a new catalytic function. It is also expected that the principal determinants of substrate specificity will be identified, which will facilitate subsequent work aimed at engineering Sbi_00515 and Swit_4259 to produce alpha-keto acids with tailored side chains. These outcomes will have a positive impact by expanding and helping to define the burgeoning ADCSF field, as well as improving the ability of researchers to more accurately predict the functions of ADCSF enzymes identified by genome sequencing.Broader Impacts. This project will use a number of strategies to promote teaching, training and learning. First, the PI's group has an established record of including undergraduates in research projects. The structural enzymology work proposed is readily divided into semester-long sub-projects that allow undergraduates to make significant contributions to the progress of the research. The PI will also incorporate actual research data into the two courses he teaches each year, both of which are primarily undergraduate offerings. These data will be used to illustrate principles of data interpretation, as well as fundamental aspects of protein structure and function. Particular attention will be paid to the overall development of the graduate students and postdoctoral scholars funded by this proposal, not only as researchers, but as fully-integrated members of the scientific community. In order to achieve this goal, all trainees will receive instruction in both written and oral presentation of research results, academic integrity, and professional development. To maximize the number of students who will benefit from the proposed research, the PI is participating in the "Students Modeling a Research Topic" (SMART) program offered through the Milwaukee School of Engineering. SMART Teams introduce 10th-12th graders to structural biology as they work in collaboration with their teacher and faculty research mentor. Finally, the PI will disseminate the results of the research not only to the scientific community, but also the general public through a series of podcasts. Thus, the research will have positive social impact, applied benefits, and advance the ADCSF field.