Sequence alignment of MLCE and FLCE together with mutated sites for mutant recombinant LCEs. (A) Sequence alignment of MLCE, FLCE, FLCE_mu20, and FLCE_mu10. Residues conserved between MLCE and FLCE are boxed with black lines. Gray boxes indicate active site consensus sequences. Red bars above the sequences indicate amino acid residues facing the active site cleft of MLCE. Amino acid residues that were mutated in FLCE_mu20 and FLCE_mu10 are also shown. The numbers and alphabetical subscripts (a–e) at the bottom of the sequences indicate two amino acid residues, which were returned to Fundulus-type mutations and introduced into FLCE_mu8a to mu8e. Light blue boxes indicate the mutated positions in FLCE_mu4. The amino acid residues of mutated sites in FLCE_mu20 (B), FLCE_mu10 (C), and FLCE_mu4 (D) are colored red, and the zinc atom at the catalytic center is colored yellow on the crystal structure of MLCE, shown as a surface representation.