Unnatural Metal-Free DNA Cutting Enzymes Using Halogenated Amino Acids

At a Glance

Researchers at Colorado State University have expanded the repertoire of biological catalysts by showing that a halogen bond (X-bond) can functionally replace the magnesium (Mg2+) cofactor in endonucleases. By mutating a metal coordinating amino acid to a meta-halotyrosine this resulted in a construct that is both acid and base catalyze.

This new class of enzymes utilizes a previously unexplored unnatural catalyst in its active site—a catalytic X-bonding enzyme, or cX-Zyme—by controverting what constitutes a metal catalyst in biochemistry.

Background

A fundamental concept in chemistry is that the properties of elements are periodic. Magnesium is a common alkaline Earth element metal cofactor in enzymes that catalyzes many biochemical reactions, including DNA processing. By replacing the catalytic magnesium in a DNA endonuclease with iodine or chlorine (group VII elements), this unnatural amino acid forms a hydrogen bond enhanced halogen bond to render the DNA backbone susceptible to hydrolysis, mechanistically analogous to but different from mangesium. This unique catalytic center opens the field of synthetic molecular biology to the design of novel enzyme active sites and creates new opportunities for metal free catalysts to be used across biology.

Overview

In these new catalysts, the metal coordinating glutamate E136 in a mouse endoculeaseG is modified to meta-halotyrosine (mXY, X = chlorine or iodine) to form a mXY-mEndoG construct that is both acid and base catalyzed. Under basic conditions, the enzyme is inactivated by standard laboratory ethylene diamine tetraacetic acid (EDTA), indicating that the halogen substituent facilitates deprotonation of the tyrosyl hydroxyl group, allowing recruitment of magnesium to restore the metal-dependent catalytic center. At low pHs, the mXY-mEndoG is resistant to EDTA inactivation and that the iodinated constructed is significantly more active than the chlorinated analogue.

These results confirm that a hydrogen bond (H-bond) enhanced X-bond as the catalyst in the mXY-mEndoG, with asparagine N103 serving as the H-bond donor that communicates the protonation state of histidine H104 to the halogen. This model is supported by mutation studies and electrostatic potential (ESP) calculations on models for the protonated and unprotonated mXY···N103···H104 system compared to the Mg2+ coordination complex of the wild type.

Figure 1: Structure and catalytic activities of mEndoG constructs. (A) Crystal structure of the mEndoG homodimer (separate monomers colored cyan and green), with the T5 DNA from the C. elegans CPS6 ortholog of EndoG docked into the active site of the green monomer. (B) Schematic for the mechanism of mEndoG, showing glutamate E136 coordinated to the catalytic Mg2+ and histidine H97 serving as a general base to deprotonate the nucleophilic water. (C) Active site in WT-mEndoG shown with Mg2+ coordinated (dashed lines) to the carboxylate oxygens of E136 through bridging waters and the carbonyl oxygen of asparagine N128. d. Model of the E136 replaced by mIY136, generating the mIY-mEndoG construct. This RotA model positions the iodine to potentially form X-bonds to the scissile phosphate oxygens of the DNA. Asparagine N103 is shown as an H-bond donor to bridge mIY136 to the protonated histidine H104. (E) The RotB model of mIY-mEndoG construct positions the iodine away from the DNA phosphate oxygens and N103.

Figure 2: Quantitation of cleaved DNA products after 30 min reaction from halogenated (mClY-mEndoG, green bars, and mIY-mEndoG, magenta bars) and unhalogenated (gray bars) tyrosine mutant constructs relative to those from WT-mEndoG at pHs from 7.0 to 9.0.

Benefits

Metal free enzyme construction for use in low pH ranges
Custom catalysts
Broad range and use of novel catalysts including Cas9 and other molecular guides
Non-natural enzymes with biological application

Applications

Cutting of nucleic acids
Custom design of enzymes previously not attainable

Publications

Ho, P. Shing et al. (2025). Design of a halogen bond-catalyzed DNA endonuclease. PNAS. https://www.pnas.org/doi/10.1073/pnas.2500099122

Last Updated: June 2025

Opportunity

Available for Exclusive Licensing
TRL: 5

IP Status

US Provisional Patent

Inventors

Pui Shing Ho
Anthony Rappe
Alexander Ho
Cesar Mendez
Margaret Walker
Ryan Czarny

Reference Number

2024-070

Licensing Manager

Steve Foster
Steve.Foster@colostate.edu
970-491-7100