Engineering Water Capture in Terpene Synthases

Enzymes are remarkable biomolecules that are able to make complicated chemical reactions occur under the mild conditions found in living systems, with remarkable precision. Terpene synthases are enzymes that catalyze the conversion of only a small number of substrates to a myriad of different hydrocarbon products called terpenoids, structures that form the chemical basis of the largest group of natural products. Many terpenoids have beneficial applications, such as the anti-cancer drug taxol, the anti-malaria drug artemisinin and so-called semiochemicals that can act as insect repellents. These are based on sesquiterpenes, a subset of the terpenoid family that contain 15 carbon atoms and derive from the same molecule, farnesyl diphosphate (FDP). Natural sesquiterpene synthases can generate more than 300 different hydrocarbon scaffolds from FDP. Whether or not these scaffolds incorporate water (through hydroxylation, or 'water capture') affects their biological activity and use. Structural and mechanistic work performed over the last decades has revealed that these enzymes share a three-dimensional fold and use similar chemical strategies to achieve the transformation of FDP into products. While much progress has been made to decipher the biochemical details, our knowledge is not complete enough to have predictive power so that a rational approach could be used to convert the 'water capture' behaviour of terpene synthases in a targeted fashion.
The proposed research, which is based on a solid foundation of previous work and the complimentary experience of the applicants, will bring together enzymologists, computational biochemists and structural biologists to generate in-depth understanding of the mechanisms of 'water capture' in terpene synthases. This understanding will then be used to rationally change 'water capture' behaviour in example enzymes, which will lead to novel terpenoid products with potential beneficial properties. The combination of state-of-the-art computational and experimental work is crucial for achieving this. By using experimental data, such as structures obtained through X-ray crystallography, computational modelling offers unique detail not accessible through laboratory experiments, which in turn can be used to predict the effect of amino acid changes. These predictions subsequently need experimental validation. Through this combined approach we will discover in great detail how enzyme structure and mobility affect the reaction outcome. Once successful alteration of water capture behaviour has been achieved, we will develop general, streamlined protocols that can be applied to other terpene synthases. These protocols will therefore allow rational modification of efficient biocatalysts to obtain specific terpenoid products with desired properties, e.g. to develop new drugs or insect repellents.

The main aim of the proposed work is to rationally change sesquiterpene synthases so that they specifically generate hydroxylated or non-hydroxylated products, different from their natural function. A combined computational/experimental approach will be used to gain in-depth understanding of the mechanisms of water binding, water flow and hydroxylation. This understanding will be used for a) targeted modification of enzyme active site and entrance loops to alter hydroxylation behaviour in example cases and b) developing step-by-step computational/experimental protocols to modify hydroxylation behaviour in terpene synthases. To gain understanding, we will use simulation and experiment to predict and verify water binding, the active enzyme-substrate complex and the detailed mechanism of key reaction steps (in particular hydroxylation by water molecules). The combined approach requires intensive collaboration between the two sites involved, as well as collaboration with overseas experts. Experimental methods will include protein modification using site-directed mutagenesis and incorporation of non-natural amino acids using a native chemical ligation approach, organic synthesis of mechanism-based inhibitors, kinetic characterization of enzymes, measurement of product distributions and product characterisation, and protein X-ray crystallography. Computational methods will include hybrid quantum mechanical/molecular mechanical simulations of reactions, molecular dynamics simulations of enzyme dynamics and water flow, and docking/Monte-Carlo protocols to predict water binding sites/affinities. By combining results from all methods, we will build up extensive and uniquely detailed knowledge on the mechanisms guiding water capture, leading to general streamlined protocols for rationally changing water capture in terpene synthases. This will then help to unlock the great potential of terpenoids as useful compounds with applications in health care, agriculture and bio-energy.

The proposed work will lead to 1) detailed, fundamental insights into terpene synthases that can be used as biocatalysts, 2) engineered enzymes for the production of new terpenoid compounds and 3) the development of protocols to rationally engineer product outcomes in other terpene synthases. The new examples of engineered biocatalysts and the methods to obtain them will be of high interest to the biotech industry, due to their ability to enhance the cost-effectiveness and sustainability of synthesising terpenoid compounds. As part of the work proposed, computational and experimental protocols will be developed that are useful for enzyme and protein engineering as well as structure-based drug design, and will thus benefit academic and industrial researchers in these areas. The work exemplifies a rational, synthetic biology approach to obtain desired biocatalysts for industrial biotechnology, and as such offers the opportunity to engage with the general public. Via outreach activities, the general public will benefit by obtaining insight into the use of synthetic biology and how publicly funded research in academia affects biotechnology, topics of interest to many.
Apart from these short-term benefits to a wide range of researchers and the public, the proposed work has significant potential impact in the medium and long term.
In the medium term, the knowledge and protocols obtained in this research can be applied to redesign natural terpene synthases into efficient biocatalysts for the production of specific desired terpenoids. This can have a significant impact by making industrial production of terpenoids efficient, sustainable and relatively cheap. By disseminating the results from our research directly to relevant biotechnological companies in the UK, this will give UK biotech a competitive edge over other biotech companies world-wide. UK-based companies are active in the field of biocatalyst optimization and bio-based chemical production, and the national economy can therefore benefit significantly.
In the long term, the application of novel terpenoid biocatalysts will allow new, beneficial terpenoid compounds to be conceived and produced. This in turn can lead to new valuable (agro)chemicals and potential new drugs, leading to healthcare and food production benefits. In addition, efficient, sustainable production of terpenoids will strengthen the UK economy whilst limiting environmental impact, thus benefitting both the general public and the UK industry.

Finally, the proposal will help bring together and integrate further two research communities that are strong within the
UK: biomolecular simulation and chemical biology/enzymology. The UK should capitalise on the strengths of these communities to catch up and, in future, overtake efforts in other countries in the area of biocatalyst (re)design. The close collaboration between computational and experimental groups, and the training and knowledge exchange activities planned, will help train a new generation of researchers. These researchers will be well-equipped for many areas that require a cross-disciplinary outlook (both in academia and industry), such as synthetic biology and industrial biotechnology. The related professional skills acquired by the researchers involved (significant IT, communication, analytical thinking and time management skills) will make them valuable for many/all employment sectors. This impact on UK 'human capital' will further contribute to the mid- to long term benefits.