Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class
Fig 2
Characterisation of protein cluster with high sequence identity to previously characterised S-2-hydroxyacid oxidases.
(A) Activity screen and protein characteristics. Dendrogram indicates protein relatedness. Superkingdoms: light purple—Bacteria, brown—Eukaryotes. Recorded activities are marked with squares, for proteins active with more than one substrate, the substrate preference is shaded with the highest activity for each enzyme scaled to 100%. Listed amino acids correspond to conserved residues in a glycolate oxidase from S. oleracea. The cartoons represent predicted domain and motif composition of the sequences, based on Pfam search. Domains lacking full Pfam alignment are represented with a sharp edge. FMN-binding domain (FMN_dh, PF01070) is marked in magenta, cytochrome b5-like heme binding domain (Cyt_B5, PF00173) is marked in green, and a prolonged stretch in loop4 is marked in blue. (B) Conserved amino acids of the active site of S-2-hydroxyacid oxidase mapped on a structure of glycolate oxidase from S. oleracea (PDB: 1GOX). Conserved residues are marked in blue, the FMN cofactor is marked in yellow, and the glycolate substrate in green. (C) Superimposed structures of the representatives of FMN-dependant 2-hydroxyacid oxidase/dehydrogenase family with their distinct motifs represented in a cartoon form: glycolate oxidase (magenta, PDB 1GOX), flavocytochrome b2 (green, PDB 1FCB), mandelate dehydrogenase (light blue, PDB 6BFG), lactate 2-monooxygenase (dark blue, PDB 6DVH).