Fig. 1. Functional evolutionary history of MPH gene family.

a Phylogenetic reconstruction of MPH family and its DHCH relatives. The catalytic activities (kcat/KM) of the enzymes for dihydrocoumarin and methylparathion are displayed in bar graphs (error bars show standard deviation). The five key mutations between the ancestral DHCH enzyme and MPH are labeled in orange on the branch between MPH-m5 and the derived MPH. The schematic phylogenetic was constructed using previously published phylogenetic reconstruction¹¹. b Overlay of the cartoon representations of the crystal structures of DHCH (cyan, PDB ID: 6c2c) and MPH (orange, PDB ID: 1p9e). The five key mutations are highlighted as sticks and labeled in orange. The two active site metal ions are shown as spheres. Residues involved in coordinating the active site metal ions are highlighted as sticks and labeled in gray. The docking pose of the methyl-parathion substrate is shown as sticks. c A cropped multiple sequence alignment of representative sequences of extant MPH, DHCH, and resurrected ancestral enzymes. Residues at the positions where the five active site mutations have occurred between DHCH and MPH are highlighted in orange. c is adapted from Yang et al.¹¹.