Catalytic halogen bonds in enzymatic bond breaking and making in DNA

Colorado State University, Fort Collins CO

Investigators

Abstract

With this award, the Chemistry of Life Processes Program of the Chemistry Division is funding Professors Pui S. Ho and Anthony K. Rappé at Colorado State University to determine how halogens can replace magnesium in the design of catalytic proteins (enzymes) that break and make the bonds in DNA. Magnesium is the metal typically used by enzymes to help cut or join DNA in cells. Dr. Ho’s group has shown that the catalytic magnesium in an enzyme found in mice can be replaced by an unnatural chlorinated or iodinated amino acid. This project will determine how an interaction called halogen-bonding allows elements on opposite ends of the periodic table to catalyze the same DNA-modifying reactions. This science probes the idea of using halogen-bonding as a design tool to engineer synthetic enzymes for biotechnology applications. This project will provide training in structural and computational chemistry to graduate and undergraduate students, including those from underrepresented groups. Finally, an outreach project will help middle school and high school students learn how scientists use “X-ray vision” see the atoms in three dimensions in DNA and proteins. The making and breaking of the bonds along the DNA backbone are critical reactions in a broad range of genomic processes. Endonucleases and ligases typically use magnesium ion or similar metal cations as catalysts to specifically cut and rejoin DNA fragments. The Ho and Rappé groups have shown that the essential catalytic divalent magnesium ion in mouse endonuclease G (mEndoG) can be functionally replaced by a chlorinated or iodinated tyrosine. The resulting halogenated construct recapitulates the activity of the wild type mEndoG to specifically cleave four-stranded DNA Holliday junctions that contain a 5-hydroxymethylcytosine base. This cleavage reaction is catalyzed by a hydrogen bond (H-bond) enhanced halogen bond (X-bond), or HBeXB, for short. In addition, the halogenated enzyme was found to catalyze a unique ATP-independent DNA joining reaction that is also seen in the wild type mEndoG. This research project will characterize the structural and mechanistic properties that allow this metal-free, X-bond catalyzed enzyme (or cX-Zyme) to act as a site-specific endonuclease and an ATP-independent ligase. From a broad scientific perspective, this work raises the interesting possibility that one may be able to exploit X-bonding quite generally as a novel method to promote specific catalytic chemistry in enzyme active sites. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.