Heme biosynthesis in mammalian systems: Evidence of a schiff base linkage between the pyridoxal 5-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase

Gloria C. Ferreira 1 3 *, Peter J. Neame 4 2, Harry A. Dailey 7 5 6

1Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612 2Institute for Biomolecular Science, University of South Florida, Tampa, Florida 33612 3H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 4Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612 5Institute for Biomolecular Science, University of South Florida, Tampa, Florida 33612 6Shriners Hospital for Crippled Children, University of South Florida, Tampa, Florida 33612 7Department of Microbiology and the Center for Metalloenzyme Studies, The University of Georgia, Athens, Georgia 30602

^*Correspondence to Gloria C. Ferreira, Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612

5-aminolevulinate synthase • heme biosynthesis • pyridoxyllysine • pyridoxal 5-phosphate

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5-phosphate-dependent enzyme and is functional as a homodimer.

The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain.

Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.