Project Summary
Biological methylation plays an integral role in human disease by regulating pathways important
for homeostasis, disease prevention and in some scenarios, can even promote disease. For example, in
bacterial ribosome systems, seemingly simple methylations can result in the development of antibiotic
resistance to current antibiotics. For a long time, methylations were known to be completed by an SN2 transfer
of the methyl group from S-Adenosyl methionine (SAM) to a nucleophilic heteroatom. Then, about two
decades ago, it was discovered that iron-sulfur [4Fe-4S] clusters had the ability to promote radical cleavage
of SAM and form 5’-deoxyadenosyl 5’-radical (5’dA•), which can promote radical methylation of
previously unactivated carbon centers! Sequencing capabilities have highlighted the vast presence of these
radical SAM methylases (RSMs) in biology, but it is still unknown what unique chemistries these enzymes are
capable of, and more importantly, how RSMs contribute to human health.
The Booker lab at Penn State has been working on characterizing RSMs that are dependent
on cobalamin (Cbl) as an intermediate methyl carrier during the methylation reaction. Recently, we have
discovered a novel subgroup of Cbl-dependent RSMs that contain an atypical Cbl-binding protein
domain. Previously unannotated, and referred to as a domain of unknown function (DUF512), this domain
differs from the canonical Cbl-binding domains, as it is C-terminal to the RS motif. Using bioinformatics, we
have identified ~4000 proteins that contain DUF512, including one from the important human pathogen
Clostridioides difficile. We expect the C. difficile DUF512 (cdDUF512) enzyme to perform a radical based
methylation on an unactivated carbon center of a protein substrate. Preliminary work, has shown that
cdDUF512 is connected to ribosome maturation, and we hypothesize that cdDUF512 methylates EngA, an
essential GTPase that stabilizes the 50S subunit of the ribosome. Ribosomal protein synthesis is a
significant antibiotic target, thus highlighting the importance of the proposed work as we uncover cdDU512’s
mechanism in C. difficile.
We propose the investigation of cdDUF512, to decipher the structural importance of the novel DUF512
domain, identify the biological substrate of these enzymes, and understand the biological importance in
C. difficile, while creating a roadmap for future RSM annotation. First, the x-ray crystal structure for cdDUF512
will be solved to decipher its important binding characteristics. Second, we will examine cdDUF512’s
connection to ribosome maturation with a combined biochemical and proteomic approach. Lastly, we will use a
radiolabeling technique, which was developed in the Booker lab, to track the methylation in cellular lysate
to identify the biological substrate. In all, we hope to progress the field of radical SAM chemistry forward,
while deciphering mechanisms that will contribute to future antibiotic development.
