Kinetic parameters of reconstructed ancestral enzymes and modern AlmA and ALAS. (A) Phylogenetic tree based on the amino acid sequences of the AlmA homologues and ALAS. Accession numbers and class of the host of each sequence are summarized in supplementary Table S1, Supplementary Material online. The organism orders are described by two letters, and the abbreviations are shown on the right side. ALAS from α-proteobacteria are shown in blue, and AlmA are shown in orange. Cyclic ALAS (cALAS) sequences are used as the out-group. Reconstructed ancestral nodes are indicated by red arrows, and AlmA from A012304 is indicated by a black arrow. (B) Kinetic parameters of AlmA, RcALAS and reconstructed enzymes as determined by the Michaelis–Menten equation. All enzymes were his-tagged on the N-terminus, and detailed conditions of the enzymatic assay are described in the Materials and Methods section. (C) Graphical representation of k_cat/K_m values of each enzyme. Error bars show the standard deviation calculated from at least three experiments. (D) Complementation or growth suppression of MGHA01 by reconstructed genes. Cells grown in L medium were washed with saline and spotted after dilution on an M9 agar plate (left) and an M9 agar plate supplemented with 5 μg/mL ALA (right).