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

Abstract

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 Å resolution (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 Å resolution. 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.

Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine

“…Because both l -homoserine and l -alanine could be converted to LCBs by SPT ( 22 ), the dissociation constants for enantiomers of these amino acids were also examined by the titration experiment. The K d value for d -homoserine was 90 ± 8 mM, and no significant spectral changes were observed up to 170 mM of d -alanine.…”

“…SPT was crystallized by the sitting drop vapor diffusion method in 24-well plates at 20 °C as described previously ( 22 , 23 ). Briefly, an aliquot of 2 μl of 20 mg/ml protein solution was mixed with 4 μl of the reservoir solution containing 100 mM Tris–HCl (pH 8.5), 200 mM sodium acetate, and 19 to 24% (w/v) PEG4000.…”

“…Bacterial SPTs from sphingolipid-containing bacteria such as Sphingomonas paucimobilis , which is rich in glycosphingolipids, and Sphingobacterium multivorum , which is rich in phosphosphingolipids, function as a water-soluble homodimer enzyme and are useful as a prototypical model system of human SPT ( 15 , 16 ). In addition to the high-resolution X-ray crystal structures of microbial SPTs ( 17 , 18 , 19 , 20 , 21 , 22 , 23 ), the structures of eukaryote SPT complexes were recently determined by cryo-EM at a resolution range of 2.6 to 3.8 Å ( 24 , 25 , 26 ). The active site architecture of the human SPT thus determined was indicated to be almost the same as that of the bacterial enzymes.…”

“…The imidazole ring of His263 stacks to the PLP pyridine ring in parallel, and Ala346 forms a van der Waals interaction with the other side of the pyridine ring of PLP. All of these amino acid residues are fully conserved in the active site of bacterial SPTs ( 17 , 22 , 23 ).…”

“…On the other hand, we previously reported that S . multivorum SPT could metabolize not only glycine and l -alanine but also l -homoserine, which is bulkier than l -serine ( 23 ), into the corresponding LCB products and form the external aldimine intermediate with a variety of amino acids or amine ligands, such as tris(hydroxymethyl)aminomethane, indicating a relatively promiscuous activity of this enzyme ( 22 , 46 ). Here, we re-examined the reactivity of the S. multivorum SPT with d -serine to answer the above question and discussed the results based on the enzymatic and structural insights on SPT.…”