File S1 - A Hybrid NRPS-PKS Gene Cluster Related to the Bleomycin Family of Antitumor Antibiotics in Alteromonas macleodii Strains

Supporting Files. Table S1, List of all genes and function for predicted genes of the pAMDE1 cluster. Table S2, C domain motifs identified in the pAMDE1 gene cluster. Amino acids that differ from the classical C domain motif are shown in red. The numbers at the top are residue identifiers as described in SBSPKS server. Table S3, Gene comparison of pAMDE1 to BLM, TLM and ZBM clusters. Table S4, C domain motifs of pAMDE1 in comparison to the BLM, TLM and ZBM motifs. Amino acids that differ from the classical C domain motif are shown in red. The numbers at the top are residue identifiers as described in SBSPKS server. Table S5, A domain motifs and amino acid specificity of pAMDE1 in comparison to that of BLM, TLM and ZBM. Amino acids that differ from the BLM, TLM and ZBM domain motifs are shown in red. The numbers at the top are residue identifiers as described in NRPSpredictor2 and SBSPKS server. Figure S1, phylogenetic tree of C domains. All known categories of C domains are shown. C domains of pAMDE1 are highlighted in bold. LCL: catalyzes peptide bond formation between 2 L-amino acids, DCL: catalyzes peptide bond formation between a D- and L- amino acid, CYC: heterocyclization domain, epimerization: flips chirality of last amino acid, dual E/C: catalyze both epimerization and condensation, modAA: modify the incorporated amino acids, H: hybrid, UNC: unclassified. Bootstrap values are shown on the branches. Figure S2, bleomycin family of antitumor antibiotics. A) Structure of BLM, TLM and ZBM. Structural differences are highlighted with red arrows. B) Gene cluster representation and protein domains of bleomycin (blm), tallysomycin (tlm) and zorbamycin (zbm). The protein domains are shown inside the boxes. The modules (NRPS-0 to NRPS-9) described for the bleomycin compounds are represented by the black lines above the domains. The NRPS genes are represented by red arrows, and the PKS gene is represented by a blue arrow. Figure S3, Cluster comparison of pAMDE1 to BLM, TLM and ZBM. All genes are colored according to the inferred function. Although all-versus-all comparisons (using BLASTn) were made, only selected pairwise comparisons are shown for clarity. The level of similarity between different contigs is indicated in the legend on the left. The name of the genes of pAMDE1 (represented by ORFs) and zorbamycin are shown. Figure S4, Structural alignment. Acyltransferase domains from four sequences of the bleomycin family and three known protein structures (PDB codes: 1MLA, 3QAT, 2JFD) are shown in the alignment. The alignment was created using the PROMALS3D web server. The first line in each block shows conservation indices for positions with a conservation index above 2. The last two lines show consensus amino acid sequence (Consensus_aa) and consensus predicted secondary structures (Consensus_ss). Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic: l; aromatic: @; hydrophobic: h; alcohol: o; polar residues: p; tiny: t; small: s; bulky residues: b; positively charged: +; negatively charged: -; charged: c. Known active site residues in the protein structure of 1MLA are indicated by a orange star symbol on top. (PDF)