Biocatalysis in academic and industrial research is a rapidly growing field for the efficient enantio and regioselective conversion of molecules under mild reaction conditions. These developments are supported by new revolutionary methods in enzyme engineering and directed evolution, powerful tools that enable researchers to customize Nature's catalysts to their substrates and environmental requirements. Among the functionally most versatile and intensely studied biocatalysts are members of the a/B hydrolase-fold family. Our laboratory recently demonstrated that the catalytic performance of one family member, the lipase B from Candida antarctica (CALB), can be significantly enhanced by using an unconventional protein engineering approach called circular permutation (CP). However, the predictive framework for the structural and functional consequences of this engineering method is very limited. This research will address two fundamental aspects of CP. Firstly, we will investigate the impact of circular permutation on protein structure with a particular focus on the new termini regions. Working with our existing permuted CALBs, we will apply x-ray crystallography, spectroscopic techniques and protein engineering to elucidate the local conformational preferences in the N and C-terminus of engineered lipases and assess their relevance to enzyme function. Secondly, we hypothesize that CP is a general method for engineering members of the a/B-hydrolase fold, due to the family's modular design, which places the catalytic residues in the structurally conserved protein core while substrate binding is determined by an interchangeable cap domain. We will test CP's broader applicability for the improvement of other a/B hydrolase-fold family members on the epoxide hydrolase from Agrobacterium radiobacter (EchA), an important biocatalyst for the preparation of asymmetric diols.
The project will have an impact on the educational infrastructure by providing an excellent training and mentoring opportunity for future scientists on all levels (undergraduate, graduate, and postdoctoral), including women and underrepresented minorities. Situated at the interface of chemistry and biology, the outlined work requires interdisciplinary collaborations between the fields of molecular biology, biochemistry, physical chemistry and organic synthetic chemistry. Each members of the research team will work on an independent yet related problem, instilling the sense of ownership but also encouraging and requiring regular communication, a process that will be facilitate informally as part of the daily interactions in the laboratory and more formally in presentations during weekly group meetings. Members of the research group are also encouraged to actively participate (and give presentations) in the monthly meetings and seminars of the Center for Fundamental and Applied Evolution (FAME). FAME represents an Atlanta-wide group of 12 PIs and their students (approx. 90 people) from the chemistry, biology, biochemistry, and chemical engineering departments at Emory University, Morehouse College, and the Georgia Institute of Technology with a common interest in biocatalysis, directed molecular evolution and combinatorial chemistry. Finally, the results for this research project will be disseminated through student presentations at local and national scientific meetings. Attendance of these events will also introduce them to the broader scientific community and new research areas.
