Effects of site-specific mutations at the 82nd and 86th amino acid residues in AlmA on the complementation ability of MGHA01. (A) Sequence alignment of AlmA and ALAS from various species. The genus abbreviations are St: Streptomyces, Rb: Rhodobacter, Rp: Rhodopseudomonas, Sa: Saccharomyces and H: Homo. The residues corresponding to 82nd and 86th of AlmA are highlighted in green. Asterisks represent conserved residues. (B) Comparison of active sites between ALAS from Rb. capsulatus (PDB ID: 2BWP) and AlmA. 3D structure of AlmA was predicted by SWISS-MODEL using ALAS from Rb. capsulatus as a template. The shortest distance between the 82nd residue and the substrate glycine is shown by arrows. (C) Complementation of MGHA01 by AlmA variants. Cells grown in L medium were washed with saline and spotted after dilution on an M9 agar plate. (D) Specific activities of His-AlmA and its variants. Specific activity of His-AlmA against serine was measured using 0.01 μM enzyme and the others with 2 μM.