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

University Of Wisconsin-Milwaukee, Milwaukee WI

Investigators

Abstract

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.