Genetically Engineered Boron-Containing Enzyme with Organocatalytic Properties for Kinetic Resolution of Hydroxyketones

Core Concepts

Genetic-code expansion can be used to create evolvable enantioselective enzymes with xenobiotic catalytic moieties like boronic acids, enabling reaction mechanisms beyond the capabilities of natural or engineered biocatalysts.

Abstract

The content discusses the creation of a genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts. This boron enzyme catalyzes the kinetic resolution of hydroxyketones by oxime formation, where interactions with the protein scaffold assist in the catalysis.
A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS), and 11B NMR spectroscopy.
The study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.

Stats

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production.
The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis.
The boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation.
A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates.

Quotes

"The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis."
"Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes."

Key Insights Distilled From

Boron catalysis in a designer enzyme - Nature

by Lars Longwit... at www.nature.com 05-08-2024

https://www.nature.com/articles/s41586-024-07391-3

Deeper Inquiries

How can the principles of genetic-code expansion be applied to create other types of designer enzymes with novel catalytic capabilities?

Genetic-code expansion involves incorporating non-canonical amino acids into proteins by reassigning codons to these amino acids. This technique can be utilized to introduce novel catalytic functionalities into enzymes, expanding their catalytic repertoire beyond what is naturally possible. By encoding specific non-biological functional groups, such as boronic acids, into the protein sequence, designer enzymes with unique catalytic capabilities can be created. Through rational design or directed evolution, these enzymes can be optimized for specific reactions, leading to enhanced catalytic efficiency and selectivity. Additionally, the use of genetic-code expansion allows for the precise control and modulation of enzyme activity, enabling the development of tailored biocatalysts for a wide range of chemical transformations.

What are the potential limitations or challenges in engineering enzymes with xenobiotic catalytic moieties like boronic acids?

While the incorporation of xenobiotic catalytic moieties like boronic acids into enzymes offers exciting possibilities for expanding the scope of biocatalysis, there are several limitations and challenges that need to be addressed. One major concern is the stability and compatibility of the engineered enzymes with the non-biological functional groups. Boronic acids, for example, may be prone to hydrolysis or other chemical modifications under certain conditions, affecting the overall catalytic activity of the enzyme. Additionally, the introduction of xenobiotic moieties could alter the structural integrity or folding of the enzyme, leading to decreased stability or activity. Furthermore, the optimization of enzyme-substrate interactions and the fine-tuning of catalytic mechanisms for xenobiotic-containing enzymes may require extensive engineering efforts and screening processes. Balancing the benefits of incorporating non-biological functionalities with the challenges of maintaining enzyme stability and activity poses a significant hurdle in the development of designer enzymes with xenobiotic catalytic moieties.

What other types of non-biological functional groups could be incorporated into enzymes to expand their reaction repertoire, and what potential applications could they enable?

In addition to boronic acids, a variety of other non-biological functional groups can be incorporated into enzymes to expand their reaction repertoire and enable new applications in biocatalysis. For example, metal complexes, such as transition metal ions or metalloporphyrins, can be introduced into enzymes to catalyze redox reactions or other metal-dependent transformations. Covalently attached organic dyes or photoactive groups can enable light-driven enzymatic reactions, opening up possibilities for photobiocatalysis. Furthermore, the integration of organometallic complexes or organocatalysts into enzymes can facilitate unique bond-forming reactions or asymmetric transformations. By harnessing the diverse reactivity of non-biological functional groups, designer enzymes can be tailored for specific chemical processes, including chemoenzymatic synthesis, pharmaceutical production, and environmental remediation. The incorporation of these novel functionalities into enzymes not only expands their catalytic capabilities but also offers innovative solutions for challenging chemical transformations.