Bacterial serine palmitoyltransferase: a water-soluble homodimeric prototype of the eukaryotic enzyme.
Serine palmitoyltransferase (SPT, EC 2.3.1.50) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to 3-ketodihydrosphingosine (KDS). We found that the gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity, and purified SPT to homogeneity. Unlike eukaryotic enzymes, this enzyme was a water-soluble homodimeric protein. 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 showed 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 purified recombinant-SPT showed the characteristic absorption spectrum derived from its coenzyme pyridoxal 5'-phosphate (PLP). The addition of the substrate, L-serine, caused spectral changes indicating the formation of the external aldimine intermediate. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.
Ikushiro
Hiroko
H
Department of Biochemistry, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki, Osaka 569-8686, Japan.
Hayashi
Hideyuki
H
Kagamiyama
Hiroyuki
H
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D051102:Serine C-Palmitoyltransferase; D020578:Sphingomonas / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase