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In this study, we expand the repertoire of biological catalysts by showing that a halo- gen bond (X-bond) can functionally replace the magnesium (Mg2+) cofactor in mouse endonuclease G (mEndoG). We mutated the metal coordinating glutamate E136 in mEndoG to a 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 the metal chelator ethylene diamine tetraacetic acid (EDTA), indicating that the halogen substituent facilitates deprotonation of the tyrosyl hydroxyl group, allowing recruitment of Mg2+ to restore the metal-dependent catalytic center. At low pHs, we observe that the mXY-mEndoG is resistant to EDTA inactivation and that the iodinated constructed is significantly more active than the chlorinated analogue. These results implicate 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+ coor- dination complex of the wild type. Thus, we have designed and engineered an enzyme that utilizes an unnatural catalyst in its active site—a catalytic X-bonding enzyme, or cX-Zyme—by controverting what constitutes a metal catalyst in biochemistry. 

In this study, we expand the repertoire of biological catalysts by showing that a halogen bond (X-bond) can functionally replace the magnesium (Mg2+) cofactor in mouse endonuclease G (mEndoG). We mutated the metal coordinating glutamate E136 in mEndoG to a 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 the metal chelator ethylene diamine tetraacetic acid (EDTA), indicating that the halogen substituent facilitates deprotonation of the tyrosyl hydroxyl group, allowing recruitment of Mg2+ to restore the metal-dependent catalytic center. At low pHs, we observe that the mXY-mEndoG is resistant to EDTA inactivation and that the iodinated constructed is significantly more active than the chlorinated analogue. These results implicate 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. Thus, we have designed and engineered an enzyme that utilizes an unnatural catalyst in its active site-a catalytic X-bonding enzyme, or cX-Zyme-by controverting what constitutes a metal catalyst in biochemistry. 

Abstract Classical hydrogen bonds have, for many decades, been the dominant non‐covalent interaction in the toolbox that chemists and chemical engineers have used to design and control the structures of compounds and molecular assemblies as novel materials. Recently, a set of non‐classical non‐covalent (NC−NC) interactions have emerged that exploit the properties of the Group IV, V, VI, and VII elements of the periodic table (the tetrel, pnictogen, chalcogen, and halogen bonds, respectively). Our research group has been characterizing the prevalence, geometric constraints, and structure‐function relationship specifically of the halogen bond in biological systems. We have been particularly interested in exploiting the biological halogen bonds (or BXBs) to control the structures, stabilities, and activities of biomolecules, including the DNA Holliday junction and enzymes. In this review, we first provide a set of criteria for how to determine whether BXBs or any other NC−NC interactions would have biological relevance. We then navigate the trail of studies that had led us from an initial, very biological question to our current point in the journey to establish BXBs as a tool for biomolecular engineering. Finally, we close with a perspective on future directions for this line of research.