Purification, metal cofactor, N-terminal sequence and subunit composition of a 5-aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'.
5-Aminolevulinic acid dehydratase was purified to apparent homogeneity from Scenedesmus obliquus, mutant C-2A', starting with serial affinity chromatography according to Wang et al., followed by separation on DEAE-Cellulose DE 52, TSKgel Toyopearl HW-55 and FPLC on Mono Q. The enzyme was purified 117-fold compared with the initial crude soluble enzyme preparation and showed a final specific activity of 9.17 microkat/kg protein at pH 8.2 at a total recovery of 7%. Mg2+ was determined to be the metal cofactor of the enzyme. It can, to a certain extent, be substituted by other divalent cations. From the purified enzyme the first 15 amino acids of the N-terminus could be determined, showing a moderate similarity to 5-aminolevulinic acid dehydratases from spinach, pea, Escherichia coli and yeast. The molecular mass of the native protein was determined by gel filtration to be 282+/-5 kDa. 42+/-1 kDa were ascertained for the subunit size by SDS/PAGE. These investigations, supported by electron microscopy, revealed that the enzyme from Scenedesmus consists of six subunits arranged in a six-membered ring. Additionally, there is some evidence that two of the rings form a sandwich-like complex.
Stolz
M
M
Fachbereich Biologie/Botanik, Philipps-Universität Marburg, Germany.
Dörnemann
D
D
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D000460:Chlorophyta / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D000623:Porphobilinogen Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002413:Cations, Divalent; D008667:Metalloproteins; D008670:Metals; D008345:Manganese; D000623:Porphobilinogen Synthase