Structural insights into the enzymatic mechanism of serine palmitoyltransferase from Sphingobacterium multivorum.
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 A 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 Calpha-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 Calpha-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.
Ikushiro
Hiroko
H
Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan. ikushiro@art.osaka-med.ac.jp
Islam
Mohammad Mainul
MM
Okamoto
Akihiro
A
Hoseki
Jun
J
Murakawa
Takeshi
T
Fujii
Shigeru
S
Miyahara
Ikuko
I
Hayashi
Hideyuki
H
D002384:Catalysis; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D010171:Palmitoyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D051102:Serine C-Palmitoyltransferase / Q000737:chemistry* / Q000378:metabolism; D045249:Sphingobacterium / Q000201:enzymology* / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry
C002882:ketodihydrosphingosine; D010171:Palmitoyl Coenzyme A; D012694:Serine; D051102:Serine C-Palmitoyltransferase; D013110:Sphingosine