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.
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.
Ferreira
G C
GC
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.
Neame
P J
PJ
Dailey
H A
HA
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001880:Boranes; D004912:Erythrocytes / Q000201:enzymology*; D008239:Lysine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011730:Pyridoxal / Q000031:analogs & derivatives* / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012545:Schiff Bases; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D001880:Boranes; D011994:Recombinant Proteins; D012545:Schiff Bases; C017696:epsilon-pyridoxyllysine; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D000624:5-Aminolevulinate Synthetase; D014357:Trypsin; D008239:Lysine