A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction.
Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity and purified SPT to homogeneity. This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2. The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5'-phosphate-dependent enzyme. The substrate specificity of the Sphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT shows about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme. This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.
Ikushiro
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Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan.
Hayashi
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H
Kagamiyama
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H
D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D019281:Dimerization; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D051102:Serine C-Palmitoyltransferase; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase