Evolution of Designer Enzyme Having Unnatural Catalytic Amino Acid

The remarkably high conversion rates of natural enzymes partially result from boosting the catalytic activity of a chosen few amino acid side chains through precise arrangement within the protein binding cavity. Researchers have recently shown that such modification is also possible for “designer” enzymes consisting of unnatural catalytic amino acids. In the journal Angewandte Chemie, they state that laboratory “evolution” of a designer enzyme having an aniline side chain led to alternatives with considerably higher activity.

(Image credit: Wiley)

The selectivity and speed with which enzymes in nature catalyze conversions are enviable. To catalytically enhance unnatural reactions, scientists imitate enzymes with the help of protein structures achieved by computer-aided protein design. Additional optimization is accomplished via repetition of a Darwinian cycle: 1) diversification via mutation, 2) identification of better catalysts, and 3) amplification of the more efficient enzyme alternatives. This enables for the manufacture of designer enzymes possessing very high activities.

Scientists guided by Clemens Mayer and Gerard Roelfes at the University of Groningen (the Netherlands) have presently shown that this kind of directed evolution is also a technique for enhancing the efficiency of a novel class of designer enzymes: enzymes that have an amino acid that is not exploited by nature.

Commencing with a protein from Lactococcus lactis, a bacterium used in the manufacture of dairy products such as buttermilk and cheese, the scientists synthesized a designer enzyme that has an amino acid with an abiotic aniline side chain (aminophenylalanine). Similar to free aniline, this amino acid catalyzes the reaction of aldehydes with hydroxylamines or hydrazines to make oximes or hydrazones, respectively.

To boost the enzyme’s activity, the scientists created enzyme variants containing mutations at amino acids near the aniline side chain. Screening of around 400 mutants yielded two contenders with better activity, one of which was exposed to a second evolutionary round. This led to the finding of more useful mutations. Numerous favorable mutations were integrated to form further variants to identify synergetic effects. In this manner, it was feasible to boost the enzyme’s conversion rate by a factor of 90.

The scientists highlight that, similar to natural enzymes, “this drastic increase is based on strengthening the inherent catalytic activity of the aniline side chain. We intend to use this principle to incorporate further organic catalysts as side chains in enzymes, and to use directed evolution to convert these into highly effective designer enzymes that can rapidly and efficiently carry out synthetically important reactions that would otherwise only run very slowly.”

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