PreTA-mediated metabolism of 5-Fluorouracil by intratumoral Citrobacter freundii drives chemoresistance in pancreatic cancer

AlphaFold2 structure prediction and FoldSeek-based homology analysis of PreT/PreA proteins The protein structures of E. coli PreT (UniProt: P76440) and PreA (UniProt: P25889), P. fluorescens PreT (UniProt: A0A5E7TWM2) and PreA (UniProt: A0A854XEI5), and human DPD (UniProt: Q12882) were retrieved from the AlphaFold protein structure database for subsequent homology comparison. Additionally, PreT and PreA protein sequences of C. freundii, L. reuteri, and P. putida strains were obtained from the NCBI protein database. These sequences in FASTA format were used as input for AlphaFold2 to predict their 3D structures and generate PDB files. Based on the AlphaFold2 structures of C. freundii PreT and PreA, the PreTA protein complex was constructed in PyMOL. Structural homology was evaluated using the FoldSeek online tool. Specifically, the C. freundii PreTA complex was compared with human DPD. Furthermore, the PreT and PreA protein structures of E. coli, L. reuteri, P. putida, and P. fluorescens strains were uploaded to the FoldSeek platform and compared with the PreT and PreA proteins of C. freundii strain to assess the structural similarity. The multiple sequence alignments (MSA) local distance difference test (LDDT) scores25 were used to assess the difference between local distances and predicted structures.