Cyclization specificity in terpene synthases by residue interaction networks

Enzymes are essential to life and critical for industrial biotechnology efforts aimed at producing everyday pharmaceuticals, biofuels, and fine chemicals. A complete understanding of enzyme function will enable us to fully exploit enzymes to successfully adapt to an ever changing and uncertain world. We are continually making major strides in our understanding of enzyme function through focusing on the active site where the chemical reactions occur. However, it is well documented in directed evolution experiments that outer tier mutations (regions of the enzyme structure distant from the active site) greatly enhance catalytic activity. Although outer tiers of protein structure clearly exert profound influence on enzyme function, this remains poorly understood; our current knowledge rests largely on retrospective structure-based analysis while limited investigations have been conducted.

From my own investigations of naturally evolved terpene synthases, enzymes that make diverse bioactive natural products, I discovered that outer tier mutations were essential for the specificity of a chemical reaction. Results from theses studies indicate that the outer tier 'communicates' to the active site through network connections in the protein structure, akin to computers linked through the Internet. Considering a folded protein as a network, this proposal will exploit well-developed graph theory tools, a branch of math to describe 'connectivity', to predict important outer tier residue positions from network analysis of protein structure. Further, we will apply structure-based combinatorial protein engineering collections of new enzymes that 'rewire' outer tier connections to begin to systematically interrogate our predictions. In addition, this work will produce rich data sets to advance our fundamental knowledge of enzymes and generate new 'parts' for synthetic biology and industrial biotechnology applications.

An emerging theme in our understanding of enzyme function is that outer tiers of protein structure clearly exert profound influence on enzyme function. This has been demonstrated in directed evolution studies and in naturally evolved terpene synthases (TPSs), enzymes that make diverse bioactive natural products. We previously found outer tier mutations were essential to achieve high product specificity in TPSs. Results from theses studies indicate that outer tier mutational effects are transmitted to the active site through residue interaction networks in the protein structure. We will use farnesene synthase (FS) from Artemisia annua, a linear product-producing TPS, as a model system to study the interplay of active site and outer tier mutations in the emergence and refinement of cyclization specificity. Considering a folded protein as a network, this proposal will exploit well-developed graph theory tools to predict functionally important outer tier residue positions from network analysis of protein structure. We will first biochemically characterize FS active site mutants that produce cyclic products from preliminary investigations to identify progenitor mutant enzymes with catalytically-robust activities. We will apply SCOPE to diversify predicted outer tier residues in progenitor enzymes and then distill minimal combinations of mutations that modulate cyclization specificity as measured by GC-MS analysis. These investigations will test predictions from graph theory and enable us to dissect the residue interaction networks linking outer tier and active site mutations. Therefore, this proposal will deliver new fundamental knowledge on how enzymes function as an integrated unit while generating new 'parts' for synthetic biology and industrial biotechnology applications.

Advances to our fundamental understanding of how enzymes work, how catalysis emerges from the functional integration of active site and outer tiers of protein structure (a systems based perspective), will be immediately exploitable by academic and industrial protein engineers. Developing systems-based engineering principles on natural product biosynthetic enzymes will have an immediate impact on the generation of fine chemicals for a range of human uses, while delivering advances to our fundamental knowledge of how enzymes work. Synthetic biology approaches will be absolutely critical to accomplishing the research goals laid out. More specific to the current work, plant terpene natural products are a rich source of bioactives, like the potent antimalarial compound artemisinin from Artemisia annua. This area of research will be conducted under the MET ISP at the John Innes Centre and is central to the theme "Understanding and Exploiting Plant and Microbial Metabolism." Terpenes greatly enhance the quality of everyday life and health as antibiotics, anticancer and immunosuppressive agents, to flavors and fragrances and other high value compounds. The fine chemicals industry continues to grow despite global economic downturns; the current work will contribute to UK economic competitiveness in this thriving area.

This work will directly impact industrial biotechnology, where the novel biocatalysts produced here will have immediate applications for the production of fine chemicals. For example, Allylix is a producer of fine chemicals for the flavor-fragrance industry and have developed a yeast system to produce terpenes by industrial scale fermentation. From my direct contacts, the company executives have expressed high interest in acquiring new sesquiterpene synthase enzymes for their operations (see accompanying letter of support). Zuvachem, a biotech start-up, has contacted me to express interest in my research program and the biocatalysts that the work in this proposal will generate (see accompanying letter of support). Further, I have identified and initiated contact with other academic and industrial partners in the agro industry. For example, the CNAP program at York will benefit from an expanded repertoire of A. annua-derived sesquiterpene synthases to exploit in their plant systems. Syngenta have immediate interest in the enzyme engineering aspects of the current work and the potential use of sesquiterpenes for ecological control of pests.

What will be done to be sure that they benefit from this research?

Scientific discoveries with potential commercial applications are patented through Plant Biosciences Ltd (PBL), a technology transfer company based at JIC that is jointly and equally owned by the Gatsby Charitable Foundation and the JIC. PBL is responsible for translating scientific discoveries at JIC into the public sector through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies. I will maintain contact with Allylix and Zuvachem regarding the development of novel biocatalysts from the studies proposed here. Specifically, I will arrange for meetings to discuss the new terpenes and enzyme activities that I identify to explore their market value and consider scale-up for commercialization. I will further develop relationships with CNAP and Syngenta through scheduled visits to deliver research seminars on my current work and through informal communications with key people in the respective organizations. I will also seek opportunities to give talks at industry-sponsored symposia to interact with end users and foster new partnerships. Additional interaction with other industrial partners will benefit from the Knowledge Exchange and Commercialization (KEC) activities supported by JIC Business Development (JIC BD).