Multiple sequence alignment of trypanosomatid prozymes with representative eukaryotic AdoMetDCs.
Residue positions are highlighted according to conservation: mainly hydrophobic (yellow), mainly small (gray), invariant residues in AdoMetDC enzymes that likely contribute to catalysis (black), coevolving residues that likely contribute to activation (magenta), conserved trypanosomatid prozyme/enzyme residues that stabilize the prozyme/enzyme interaction (orange), and conserved trypanosomatid prozyme/enzyme residues that form the N-helix pocket (wheat). Sequences are labeled according to NCBI GenInfo Identifier (GI) followed by the species. Alignment sections are labeled above according to taxonomy group. Non-conserved termini and insertions are removed from some sequences, with the numbers of omitted insertion residues that belong to unconserved positions indicated with brackets. Aligned sequences were ordered according to taxonomy, with the exception of protist sequences, which were split according to their similarity to other sequences in the alignment (Sphaeroforma arctica and Salpingoeca rosetta sequences are closer to metazoa than other protists) and to distinguish the Trypanosomatid group that contains prozyme sequences. Trypanosomatids were supplemented with sequences from additional species present in the NR database (Phytomonas sp. isolate EM1, Angomonas deanei, and Strigomonas culicis), and indicated prozyme sequences were extended to the N- or C-termini using TBLASTN (marked by *). The confirmed AdoMetDC domains were found to be fused to several additional domains in select sequences, including an AdoMetDC leader (pfam08132) in 35 plant sequences, an f-box-like domain (pfam12937) in one fungal sequence, pyridoxal-dependent ornithine decarboxylase (pfam02784 and pfam00278) in 9 apicomplexan and 3 plant sequences, and protein prenyltransferase alpha subunit repeat (pfam01239) in one Blastocystis sequence. The proline residue (T. brucei P31) that alters peptide bond isomerization upon activation belongs to the Trypanosomatid AdoMetDC sequence motif "FEGPEK" and is present in all complete fungal sequences, with a single exception from Vanderwaltozyma polyspora (XP_001646102.1). P31 was also present in all but 19 protist sequences, where it was replaced in all apicomplexans (11), choanoflagellates (2), Blastocystis (2), Perkinsida (1), Ichthyophonida (1), Apusomonadidae (1), and Capsaspora (1). Inspection of the multiple sequence alignment identified several residues that appeared to coevolve with the P31 in protist sequences (that have not lost the proline), including T104, N132, H172, and C269. The H172 coevolution also extends throughout fungal sequences, while the T104, N132, and C269 are restricted to a more limited subset. These residues occupy key positions in TbAdoMetDC with respect to the conformational changes that occur upon prozyme activation as described in the main manuscript.