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Journal of Biochemistry Advance Access originally published online on June 29, 2009
Journal of Biochemistry 2009 146(4):549-562; doi:10.1093/jb/mvp100
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Structural Insights into the Enzymatic Mechanism of Serine Palmitoyltransferase from Sphingobacterium multivorum

Hiroko Ikushiro1,*, Mohammad Mainul Islam1,2, Akihiro Okamoto1,3, Jun Hoseki1,4, Takeshi Murakawa1, Shigeru Fujii5, Ikuko Miyahara6 and Hideyuki Hayashi1,{dagger}

1Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan; 2Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA; 3School of High Technology for Human Welfare, Tokai University, Numazu, Shizuoka 410-0395; 4Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507; 5Laboratory of Chemistry, Kansai Medical University, Hirakata, Osaka 573-1136; and 6Department of Chemistry, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan

*To whom correspondence should be addressed. Tel: +81-72-684-7291, Fax: +81-72-684-6516, E-mail: ikushiro{at}art.osaka-med.ac.jp

{dagger}Correspondence may also be addressed. Tel: +81-72-684-6416, Fax: +81-72-684-6516, E-mail: hayashi{at}art.osaka-med.ac.jp

Received May 26, 2009; Accepted June 19, 2009


  Abstract

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyses the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of L-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The crystal structure of SPT from Sphingobacterium multivorum GTC97 complexed with L-serine was determined at 2.3 Å resolution. The electron density map showed the Schiff base formation between L-serine and PLP in the crystal. Because of the hydrogen bond formation with His138, the orientation of the C{alpha}–H bond of the PLP–L-serine aldimine was not perpendicular to the PLP–Schiff base plane. This conformation is unfavourable for the {alpha}-proton abstraction by Lys244 and the reaction is expected to stop at the PLP–L-serine aldimine. Structural modelling of the following intermediates indicated that His138 changes its hydrogen bond partner from the carboxyl group of L-serine to the carbonyl group of palmitoyl-CoA upon the binding of palmitoyl-CoA, making the L-serine C{alpha}–H bond perpendicular to the PLP–Schiff base plane. These crystal and model structures well explained the observations on bacterial SPTs that the {alpha}-deprotonation of L-serine occurs only in the presence of palmitoyl-CoA. This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction.

Key Words: enzyme reaction mechanism, PLP-dependent enzyme, protein structure, serine palmitoyltransferase, sphingolipid biosynthesis

Abbreviations: ALAS, 5-aminolevulinic acid synthase; AONS, 8-amino-7-oxononanoate synthase; AON, 8-amino-7-oxononanoate; KBL, 2-amino-3-ketobutyrate CoA ligase; KDS, 3-ketodihydrosphingosine; LCB, long chain base; PLP, pyridoxal 5'-phosphate; POAS, PLP-dependent {alpha}-oxamine synthase; SPT, serine palmitoyltransferase; spSPT, Sphingomonas paucimobilis SPT; smSPT, Sphingobacterium multivorum SPT


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