Reconstruction, enzymology and structural biology of extinct ancestral enzymes

Supervisors: Rafael da Silva, John Mitchell

Project Description:

Modern bioinformatics tools allow the accurate prediction and reconstruction of ancestral amino acid sequences for extinct enzymes. DNA synthesis permit their genes be cloned and recombinant product to be produced in bacteria. This has transformed the structural and mechanistic characterisation of extinct enzymes. This project focuses on cold- and warm-adapted (R)-3-hydroxybutyrate dehydrogenase (HBDH), an enzyme with broad applicability in biocatalysis, biotechnology, and diagnostics. The research will start with the reconstruction of ancestral sequences for extinct precursors of modern cold- and warm-adapted HBDH, going back billions of years. Selected ancestral sequences will be cloned, expressed in E. coli and the enzymes purified. The structures of the ancestral enzymes will be solved by X-ray crystallography, and their biophysical and catalytic properties interrogated by chromatography, differential scanning fluorimetry, and enzyme kinetics. These properties will be compared with modern cold- and warm-adapted HBDHs, whose structures and catalytic properties have been determined in the da Silva lab. The results will generate high-impact publications. They will also inform on the structural basis for the evolution of substrate specificity and thermal properties of these enzyme, which could be explored in biotechnology applications.

A PhD student working in this project will master skills in bioinformatics and ancestral sequence reconstruction, in molecular biology, protein chemistry, enzymology, and X-ray crystallography. He/she will also acquire proficiency in scientific writing and presentation via the preparation of scientific articles and seminars, and presentations at conferences.

He/she will also have at their disposal the full range of training and workshops available through the EASTBIO PhD program and the award-winning GradSkills at the University of St Andrews.

The successful candidate will have obtained or be about to obtain at least a 2:1 in Biochemistry, Chemistry or closely related field, and demonstrated research experience. MSc or MRes is preferable. Interested candidates should contact Dr da Silva before applying.


1.    Fisher G, Thomson CM, Stroek R, Czekster CM, Hirschi JS, da Silva RG (2018) Allosteric activation shifts the rate-limiting step in a short-form ATP phosphoribosyltransferase. Biochemistry 57, 4357-4367.
2.    Alphey MS, Fisher F,Ge Y,Gould ER, Machado TFG, Liu H, Florence GJ, Naismith JH, da Silva RG (2018) Catalytic and anticatalytic snapshots of a short-form ATP phosphoribosyltransferase. ACS Catal 8, 5601-5610.
3.    Mitchell JB (2017) Enzyme function and evolution. Curr Opin Struct Biol 47, 151-156.

To apply for this project, please go to this link.