JB Accepts, published online ahead of print on 8 June 2007

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J. Bacteriol. doi:10.1128/JB.00194-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Molecular Characterization of Membrane-Associated Soluble Serine Palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii

Hiroko Ikushiro^*, Mohammad Mainul Islam, Hiromasa Tojo, and Hideyuki Hayashi^*

Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan. Tel.: +81-72-684-7291; FAX: +81-72-684-6516; Department of Biochemistry and Molecular Biology, Osaka University, Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

^* To whom correspondence should be addressed. Email: ikushiro{at}art.osaka-med.ac.jp. hayashi{at}art.osaka-med.ac.jp.

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent -oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (Ikushiro et al., J. Biol. Chem. 276, 18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum and Bdellovibrio stolpii, respectively. Each gene product exhibits an 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by HPLC/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl-CoA concentrations higher than 100 µM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that, unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.

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