Structural insights into the substrate recognition of serine palmitoyltransferase from Sphingobacterium multivorum.

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis, which catalyzes the pyridoxal-5'-phosphate-dependent decarboxylative condensation reaction of l-serine (l-Ser) and palmitoyl-CoA (PalCoA) to form 3-ketodihydrosphingosine called long chain base (LCB). SPT is also able to metabolize l-alanine (l-Ala) and glycine (Gly), albeit with much lower efficiency. Human SPT is a membrane-bound large protein complex containing SPTLC1/SPTLC2 heterodimer as the core subunits, and it is known that mutations of the SPTLC1/SPTLC2 genes increase the formation of deoxy-type of LCBs derived from l-Ala and Gly to cause some neurodegenerative diseases. In order to study the substrate recognition of SPT, we examined the reactivity of Sphingobacterium multivorum SPT on various amino acids in the presence of PalCoA. The S. multivorum SPT could convert not only l-Ala and Gly but also l-homoserine, in addition to l-Ser, into the corresponding LCBs. Furthermore, we obtained high-quality crystals of the ligand-free form and the binary complexes with a series of amino acids, including a nonproductive amino acid, l-threonine, and determined the structures at 1.40 to 1.55 Å resolutions. The S. multivorum SPT accommodated various amino acid substrates through subtle rearrangements of the active-site amino acid residues and water molecules. It was also suggested that non-active-site residues mutated in the human SPT genes might indirectly influence the substrate specificity by affecting the hydrogen-bonding networks involving the bound substrate, water molecules, and amino acid residues in the active site of this enzyme. Collectively, our results highlight SPT structural features affecting substrate specificity for this stage of sphingolipid biosynthesis.

Crystal structure of Sphingobacterium multivorum serine palmitoyltransferase complexed with tris(hydroxymethyl)aminomethane.

Serine palmitoyltransferase (SPT) catalyses the first reaction in sphingolipid biosynthesis: the decarboxylative condensation of L-serine (L-Ser) and palmitoyl-CoA to form 3-ketodihydrosphingosine. SPT from Sphingobacterium multivorum has been isolated and its crystal structure in complex with L-Ser has been determined at 2.3 Šresolution (PDB entry 3a2b). However, the quality of the crystal was not good enough to judge the conformation of the cofactor molecule and the orientations of the side chains of the amino-acid residues in the enzyme active site. The crystal quality was improved by revision of the purification procedure and by optimization of both the crystallization procedure and the post-crystallization treatment conditions. Here, the crystal structure of SPT complexed with tris(hydroxymethyl)aminomethane (Tris), a buffer component, was determined at 1.65 Šresolution. The protein crystallized at 20°C and diffraction data were collected from the crystals to a resolution of 1.65 Å. The crystal belonged to the tetragonal space group P41212, with unit-cell parameters a = b = 61.32, c = 208.57 Å. Analysis of the crystal structure revealed C4-C5-C5A-O4P (77°) and C5-C5A-O4P-P (-143°) torsion angles in the phosphate-group moiety of the cofactor pyridoxal 5'-phosphate (PLP) that are more reasonable than those observed in the previously reported crystal structure (14° and 151°, respectively). Furthermore, the clear electron density showing a Schiff-base linkage between PLP and the bulky artificial ligand Tris indicated exceptional flexibility of the active-site cavity of this enzyme. These findings open up the possibility for further study of the detailed mechanisms of substrate recognition and catalysis by this enzyme.