Biochemical Characterization of the DUF3328 Protein in the Biosynthesis of Cyclic Peptide Cyclochlorotine

Cyclic peptide natural products are valuable sources for medicine. They exhibit significant biological and chemical diversity. Cyclochlorotine, a fungal cyclic pentapeptide produced by Talaromyces islandicus, possesses unique structural modifications, including dichlorination and hydroxylation, yet the enzymatic basis for these transformations remains poorly understood. This study biochemically characterizes the Domains of Unknown Function 3328 (DUF3328) protein family and investigates its role in cyclochlorotine biosynthesis. Through transcriptomic sequencing and CRISPR/Cas9 knockout experiments, I revealed that CctP2 is essential for chlorination and CctR is required for hydroxylation. Computational sequence and structural analyses using AlphaFold suggested that DUF3328 proteins contain a conserved HxxHC(x)nHxxHC motif, a putative metal-binding site. Structural modeling further indicated that DUF3328 proteins form disulfide-linked homodimers, an unusual feature among biosynthetic enzymes. To elucidate their biochemical roles, I purified CctR and CctP2 from Sf9 insect cells, overcoming challenges posed by their membrane association and intrinsic disorder. In vitro assays demonstrated that CctR is a copper-dependent enzyme that hydroxylates cyclochlorotine, and dimerization is essential for the activity. Mechanistic studies using isotopic labeling confirmed dioxygen as the oxygen source. Copper redox cycling was found to be essential, with Cu(I) required for the catalysis. This work establishes DUF3328 proteins as a new class of copper-dependent enzymes involved in fungal secondary metabolism. The discovery of their catalytic mechanisms expands our understanding of enzymology and provides a foundation for future enzyme characterization in this family. More broadly, this study highlights the power of computational tools such as AlphaFold in guiding the functional characterization of previously uncharacterized protein families.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2025

Author dc:creator

• Huang, Wentao

Advisor dc:contributor.advisor

• Weng, Jing-Ke

Statement dc:rights

• In Copyright - Educational Use Permitted
• Copyright retained by author(s)

Licence dc:rights.uri

• https://rightsstatements.org/page/InC-EDU/1.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/162094

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/162094