Fig. 2

Ecm14 is conserved in Ascomycota, but not in other fungal phyla. BLASTp was performed with bovine CPA1 as the query sequence and all fungal sequences as the search set. Two thousand sixty-six sequences of 150 amino acids or longer that contained the majority of the carboxypeptidase domain were identified using Clustal Omega and the best maximum likelihood phylogenetic tree inferred using IQ-TREE. Branch support was determined by 1000 ultrafast bootstrap replicates, and bootstrap support values are shown at key nodes. The resulting tree contains three predominant groups consistent with CPB-like, CPA-like, and Ecm14-like properties. The identities of these groups are demonstrated by the active site residues found in all metallocarboxypeptidases (shown in black on the right) and unique variants (red; particularly at position 255) that often define the subclass. On the phylogenetic tree, sequences from basal (monokaryotic) fungi are shown in pink. CPB sequences from ascomycetes are shown in red, with co-clustering sequences lacking the required Asp at position 255 shown in a darker red. CPA-like sequences, mostly from ascomycetes, are shown in blue. Ecm14 sequences, all from ascomycetes, are shown in dark green. A sister clade of Ecm14-like proteins from basidiomycetes, and with unique and conserved ‘active’ site residues, is shown in lighter shades of green. Ecm14 proteins are defined by the presence of N144D and E270K substitutions