Insights into enzyme selectivity open new pathways for pharmaceutical synthesis

Enzymes are the molecular tools of life. They accelerate chemical reactions and are able to distinguish very precisely between molecules that are similar to one another. The high selectivity of many enzymes is key to the production of important medicines such as penicillin and cyclosporine. But how did enzymes develop this ability to distinguish between similar chemical functionalities with such precision

An international research team, including researchers from Graz University of Technology (TU Graz), has investigated this question using a group of plant enzymes known as borneol dehydrogenases as a case study. The results show that it is not only changes in catalytic center of the enzyme, but also changes at other sites on the protein, which together can have a decisive influence on an enzyme's selectivity. The results of the study have been published in Nature Communications.

An evolutionary path to selectivity

Borneol dehydrogenases (BDHs) are plant enzymes that are involved, among other things, in the formation of camphor. Some of these enzymes are able to distinguish very specifically between the two mirror-image forms of the molecule 1-borneol. One of the two borneol variants is preferentially bound to the enzyme and is thus made available for oxidation processes. This high selectivity of the enzymes is of interest for many biotechnological applications, such as the production of active pharmaceutical ingredients, cosmetics and fragrances.

Particularly in the manufacture of medicines, the effects of mirror-image variants can vary significantly, and using the wrong mirror-image variant can cause side effects. "While natural enzymes recognise these mirror images with a high degree of precision, in many cases it is unclear why this function has become established through evolution and which changes in the enzyme were responsible for this," says Robert Kourist, head of the Institute of Molecular Biotechnology at TU Graz.

To investigate how the selectivity of BDHs has evolved, the researchers drew on sequences from 97 related enzymes from 59 plant genera and used these to reconstruct various "ancestral enzymes". Using these reconstructed proteins, they were able to trace the various stages of a possible evolutionary pathway.

This revealed a significant evolutionary leap: a particular reconstructed ancestral enzyme showed little distinction between the two mirror-image forms of the molecule, whereas a later ancestral enzyme, by contrast, was highly selective."

Jasmin Zuson, Study Co-First Author, Institute of Molecular Biotechnology

Interaction between the active site and the periphery is crucial

There are 19 amino acid changes between the two enzyme variants under closer scrutiny. Only one of the changes affects the active site. Although this mutation alone did alter the enzyme's selectivity to a measurable extent, further changes at places outside the active site were necessary to achieve very high selectivity. "The results show that the function of an enzyme cannot be explained solely on the basis of its active site. Changes in other parts of the protein can, when acting in combination, have a decisive influence on the properties of the enzyme as a whole," says Robert Kourist.

Molecular simulations provided a possible explanation for this. It is not only the sequence of the enzyme's amino acids that influences its reaction selectivity, but also its shape and mobility, which are largely determined by the periphery of the molecule. It influences how an enzyme and a reactant can come into contact with one another in a liquid. In the study, the highly selective molecular form more frequently adopted a spatial arrangement that enables a successful reaction.

Prospects for enzyme engineering

The study thus provides new insights into how evolution gives rise to molecular precision. In the long term, this knowledge may also be relevant to biotechnology: "A better understanding of the evolutionary origins of selectivity could help us to optimise enzymes more specifically for certain tasks in the future," says Robert Kourist.