The solution structure and heme binding of the presequence of murine 5-aminolevulinate synthase

Abstract

The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and 1H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H2O solutions and micellar environments. A well defined amphipathic α-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short α-helix, defined by A5–L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and 1H NMR study of hemin and psALAS indicated that a heme–peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.