Top, white bar represents the wild type (WT) ALAS sequence with highlighted amino acids previously reported to be essential for ALAS function (12, 19–21). The sequence numbering for the wild type erythroid ALAS is indicated at the top, and the amino acid numbering at the left of each variant indicates its N-terminal amino acid. Thus, for each variant the amino acid corresponding to the wild type ALAS N-terminal amino acid is represented by the junction between the black and white bars. A, active, perfectly circularly permuted ALAS variants obtained from the screening of a random library. B, rationally designed ALAS circularly permuted variants. Only A475 was found to be active, based on its ability to rescue HU227 cells on selective medium without ALA.