Acceleration of the Substrate Cα Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase

Shigeru Fujii,Hiroko Ikushiro,Hideyuki Hayashi,Yuka Shiraiwa

doi:10.1074/jbc.m706874200

Abstract

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.

Highlights

Eukaryotic Serine palmitoyltransferase (SPT) have been known to exist as membranebound heterodimers composed of two subunits called SPTLC1 (LCB1), which does not have a pyridoxal 5؅-phosphate (PLP)-binding motif, and SPTLC2 (LCB2), which carries a lysine residue that forms the Schiff base with PLP [5,6,7,8,9,10]
Generation of the Quinonoid Intermediate from the External Aldimine by S-(2-Oxoheptadecyl)-CoA—Sphingomonas SPT has absorption bands at 338 and 426 nm, which arise from the enolimine and ketoenamine tautomers, respectively, of the PLP-Lys265 Schiff base
The majority of the SPT-L-serine complex exists as the external aldimine

Summary

Introduction

Eukaryotic SPTs have been known to exist as membranebound heterodimers composed of two subunits called SPTLC1 (LCB1) (long chain base 1), which does not have a PLP-binding motif, and SPTLC2 (LCB2), which carries a lysine residue that forms the Schiff base with PLP [5,6,7,8,9,10]. In the presence of 10 mM L-serine and 5 ␮M SPT, the addition of 1.5 mM palmitoyl-CoA resulted in stoichiometric decreases in the signal intensity of the ␤-methylene proton of L-serine (data not shown), showing that the reaction completely proceeded.

Results

Conclusion