PDF(Pubmed)
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.
摘要：
丝氨酸棕榈酰转移酶（SPT）催化鞘脂生物合成中的第一个反应：L-丝氨酸（L-Ser）和棕榈酰-CoA的脱羧缩合形成3-酮二氢鞘氨醇。已分离出多活鞘杆菌属的SPT，并以2.3µA的分辨率确定了其与L-Ser复合的晶体结构（PDB条目3a2b）。然而,晶体的质量不足以判断辅因子分子的构象和酶活性位点氨基酸残基侧链的取向。通过修订纯化程序以及优化结晶程序和结晶后处理条件，可以改善晶体质量。这里,SPT与三（羟甲基）氨基甲烷（Tris）络合的晶体结构，缓冲组件，在1.65的分辨率下确定。蛋白质在20°C结晶，并从晶体中收集衍射数据，分辨率为1.65。晶体属于四方空间群P41212，晶胞参数a=b=61.32，c=208.57。晶体结构分析显示，辅因子吡哆醛5'-磷酸（PLP）的磷酸基团部分中的C4-C5-C5A-O4P（77°）和C5-C5A-O4P-P（-143°）扭转角度比先前报道的晶体结构中观察到的更合理（14°和151°，分别)。此外，清晰的电子密度显示PLP和庞大的人工配体Tris之间的希夫碱连接，表明该酶的活性位点腔具有出色的灵活性。这些发现为进一步研究该酶的底物识别和催化的详细机制开辟了可能性。
