Enzymes are biological catalysts – typically proteins – that accelerate chemical reactions. Their structure determines which molecules bind to them as well as the speed of biochemical reactions. This makes enzymes particularly valuable in biotechnology, as their properties can be harnessed to carry out specific chemical reactions. The international research team led by Dr. Bernhard Loll from the Department of Biology, Chemistry, Pharmacy at Freie Universität Berlin, Professor Robert Kourist from Graz University of Technology, Austria, and Professor Lynn Kamerlin from Georgia Institute of Technology, United States, studied a group of plant enzymes called borneol dehydrogenases. These convert the alcohol borneol into camphor in a chemical reaction. Borneol exists in two mirror-image forms called enantiomers that are comparable to a left and right hand. Some enzymes strongly prefer the formation of one form over the other, while others barely distinguish between borneol and isoborneol.
“We were able to demonstrate how the different reaction to the two different borneol forms developed over the course of the enzymes’ evolution. What was crucial here was not major structural changes to the enzyme, but the interplay of many small changes that influenced the dynamics of the enzyme and bound molecule. This provides us with important indications of how enzymes could be specifically engineered for biotechnological applications in the future,” explains Dr. Bernhard Loll from Freie Universität Berlin, one of the authors of the study.
The researchers wanted to find out how this difference emerged as these enzymes evolved. Using ancestral sequence reconstruction, the team computationally inferred the sequences of ancient enzymes from the borneol dehydrogenase family tree of ninety-seven plant sequences, reproduced them in the laboratory, and compared them.
The oldest unselective ancestor, N30, was compared with a younger ancestor, N32, which is highly selective. Between them there were nineteen mutations to individual amino acids – the building blocks of proteins – only one of which was located in the enzyme’s active site, where the chemical reaction takes place. Introducing this mutation into the unselective ancestor doubled its selectivity to the two borneol forms, and reversing it in the selective ancestor made that enzyme lose its preference.
Yet this change alone was not enough to explain selectivity. Only in combination with three additional mutations far from the active site did the ancestor N30 become highly selective to one of the two borneol forms. This meant that the researchers were able to demonstrate that even peripheral areas of an enzyme can affect the chemical reaction at the catalytic site.
The reconstructed enzymes initially showed no obvious structural differences that would explain selectivity. However, computational simulations showed that both borneol forms behaved differently in the enzyme-substrate complex.
The form that reacted better with the enzyme stayed in a catalytically productive position for longer, while the binding in the other form was held less tightly and was more exposed to the water in which it was dissolved. What was crucial here was not major structural changes to the enzyme, but the interplay of many small changes that influenced the dynamics of the enzyme and bound molecule.
The results demonstrate how the different reaction to the two different borneol forms developed over the course of the enzyme’s evolution. Using the example of an enzyme that did not distinguish much between the two forms, it was possible to show that changes to multiple regions of the protein resulted in one form being strongly preferred over the other.
Borneol and its mirror-image form isoborneol can be obtained as a mixture from α-pinene, an inexpensive bio-based raw material. Enzymes that demonstrate greater selectivity to one of the two forms could therefore help to produce individual molecule forms for use in pharmaceuticals, cosmetics, and fragrances or separate them from mixtures.
The findings suggest that enzyme engineering should look beyond the active site, because changes in peripheral regions can fine-tune selectivity and may be missed by conventional design strategies.
Nature Communications
Deciphering the evolutionary origin of the enantioselectivity of short-chain dehydrogenases from plants toward 1-borneol