Repurposing Ancient Enzymes for Modern Functions Through Simulation | Department of Chemistry

Repurposing Ancient Enzymes for Modern Functions Through Simulation

Ramalingaswami Re-entry Research Grant, Department of Biotechnology

Our objectives are to use computer simulations, in close collaboration with experiment, to:
a) Study the role of conformational flexibility in the evolution of new functions in the scaffolds provided by ancestral enzymes, using the TIM-barrel as a model scaffold.
b) Study the effect of different mutations on the thermostability and chemical versatilities of ancestral proteins, using HisA and TrpF as model systems.
c) Develop a new scoring functions to estimate the flexibility and evolvability of enzyme structure from a structural bioinformatics perspective, providing a valuable and efficient screening tool for artificial enzyme design.


Enzymes catalyze many diverse and energetically demanding biochemical reactions, with enviable rates and selectivities [1]. The scope of use of enzymes is not, however, limited to cellular functions, but they are also extensively useful as extra-celullar catalysts for example for generating new pharmaceutics or biofuels, as well as in synthetic chemistry [2]. This versatility makes them a fascinating target for chemists and biologists to design novel biocatalysts for specific chemical reactions of interest [3]. However, enzymes extracted from the natural sources are often hard to use effectively for non-biological tasks, for example due to their lack of stability under industrial conditions [4], and they need to be engineered to improve their physio-chemical properties for the process of interest. At the same time, however, despite many great advances in the field, enzyme engineering based on only simple chemical principles is also often not viable, in part due to our incomplete knowledge of protein structure function relationships [5].
Here, computational tools can play an important role in both the de novo design of new enzymes and in engineering existing enzymes towards new functionalities [6,7]. For example, such tools can aid in the identification of suitable scaffolds for the insertion of new activities, as well as in identifying key residues that can be targeted for subsequent site directed mutagenesis in the laboratory [5]. As another example, phylogenetic analysis can provide sequences of ancestral precursors to modern enzymes, which can then be generated in the laboratory based on ancestral sequence reconstruction [8]. These enzymes tend to provide highly evolvable and chemically multifunctional (i.e. catalytically promiscuous) structures, with great potential to serve as scaffolds for generating enzymes with novel functionalities [9]. In addition, the observed high stability of such ancient proteins allows for functionally useful but destabilizing mutations to be accepted without compromising the overall folding of these proteins [10]. Finally, the catalytic promiscuity of these ancient proteins has been argued to be linked with their higher conformational flexibility. Therefore, harnessing the flexibility of these ancestral scaffolds is a promising direction for the redesign of ancestral enzymes towards modern catalytic functions [11].

1. Benkovic SJ, Hammes-Schiffer S. Science, 2003, 301, 1196.
2. Schmid et al. Nature, 2001, 409, 258.
3. Schoemaker et al. Science, 2003, 299, 1694.
4. Koeller KM, Wong C-H, Nature, 2001, 409, 232.
5. Hilvert D, Annu. Rev. Biochem. 2013. 82, 447.
6. Butterfoss GL, Kuhlman B. Annu. Rev. Biophys. Biomol. Struct. 2006, 35,49.
7. Pantazes et al. Curr. Opin. Struct. Biol. 2011, 21, 467.
8. Thornton, JW. Nat. Rev. Genet. 2004, 5, 366.
9. Risso et al. J. Am. Chem. Soc. 2013, 135, 2899.
10. Wheeler et al. Curr. Opin. Struct. Biol. 38, 2016, 37.
11. Tokuriki & Tawfik, Science 2009, 324, 203.


Assistant Professor


Surajit Kalita
PhD Student
Ankita Tripathi
PhD Student
Shalini Yadav
PhD Student
Shakir Ali Siddiqui
PhD Student
Vandana Kardam
PhD Student