Minimalist Active‐Site Redesign: Teaching Old Enzymes New Tricks

Abstract

Although nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized microenvironments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity. The prediction and control of these effects provides a promising way to access new functions. The development of methods and strategies to explore the untapped catalytic potential of natural enzyme scaffolds has been pushed by the increasing demand for industrial biocatalysts. This Review describes the use of minimal modifications at enzyme active sites to expand their catalytic repertoires, including targeted mutagenesis and the addition of new reactive functionalities. Often, a novel activity can be obtained with only a single point mutation. The many successful examples of active-site engineering through minimal mutations give useful insights into enzyme evolution and open new avenues in biocatalyst research.

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

“…Rational design of enzymatic function is a challenging task and generally requires large changes in the active site: for example, the classic case of conversion of trypsin activity into that of chymotrypsin required 11 substitutions in 4 sites on the protein (30). In general, changing substrate specificity is easier than changing the reaction mechanism of an enzyme (31). The family of lysosomal glycosidases might prove to be a fruitful target for further enzyme engineering, because many glycosidases use a similar mechanism with an arrangement of two carboxylates located on opposite sides of the glycosidic linkage to be cleaved.…”

Computational design of biological catalysts

“…In this sense, despite an enzyme being generally defined as a selective catalyst capable of differentiating between different substrates and speeding up the rate of a particular chemical reaction, some enzymes have been found to present promiscuous activity, accepting alternative substrates and catalyzing secondary reactions. [2][3][4][5][6] This promiscuity provides a raw starting point for the evolution of enzymes, as a new duplicated gene presenting low activity would be the germen for adaptive evolution. 3 In fact, new enzymatic functions can evolve over a period of years or even months, as recently happened as a response to new synthetic chemicals or drugs.…”

“…6 As a consequence, the active sites of these existing enzymes provide obvious materials to engineer novel enzymes with new catalytic functions. 2 Enzyme promiscuity can be classified in three different categories: substrate promiscuity or ambiguity (the enzyme accepts structurally distinct substrates but catalyzes the same chemical reaction), catalytic promiscuity (the enzyme accepts different substrates and catalyzes different overall reactions), and product promiscuity (the enzyme accepts a single substrate and uses similar chemical mechanisms to catalyze the formation of different products). 2 An example of the substrate promiscuity can be illustrated with the cytochrome P450 (CYP), a vast superfamily of enzymes found in almost all life forms.…”

Green chemistry: the emergence of a transformative framework

“…Potentially high regio-and stereospecificity are a driving force for applying these engineered enzymes in asymmetric synthesis. Further development of non-natural activity should make biocatalysts more broadly useful (39). Directed evolution, or optimization of enzymes by mutation and screening, emerged in the 1990s as a powerful technique to produce broadly useful biocatalysts (40).…”