Gardberg Anna, Sankaran Banumathi, Davies Doug, Bhandari Janhavi, Staker Bart, Stewart Lance
Emerald BioStructures, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Sep 1;67(Pt 9):1055-9. doi: 10.1107/S1744309111021841. Epub 2011 Aug 13.
Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO(4), 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml(-1) and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C222(1), with unit-cell parameters a=121.46, b=135.82, c=61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C222(1) and the unit-cell parameters were a=121.96, b=137.61, c=62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA.
在广泛的真核生物和原核生物中都发现了果糖二磷酸醛缩酶（FBPA）。FBPA催化1,6-二磷酸果糖裂解为3-磷酸甘油醛和磷酸二羟丙酮。SSGCID已经报道了几种来自致病源的FBPA结构。对真核微孢子虫寄生虫兔脑炎微孢子虫基因组的生物信息学分析揭示了一种FBPA同源物。本文报道了该酶在天然底物FBP存在下以及与部分底物类似物磷酸盐结合时的结构。纯化后的酶在pH 6.5的90 mM双三羟甲基丙烷、18%聚乙二醇3350、18 mM磷酸氢二钠钾、10 mM尿素中结晶得到磷酸盐结合形式，在pH 6.5的100 mM双三羟甲基丙烷、20%聚乙二醇3350、20 mM 1,6-二磷酸果糖中结晶得到FBP结合形式。两种情况下蛋白质浓度均为25 mg/ml，并采用坐滴气相扩散法。对于FBP结合形式，在100 K下从单晶收集到分辨率为2.37 Å的数据集。该晶体属于正交晶系空间群C222(1)，晶胞参数a = 121.46、b = 135.82、c = 61.54 Å。结构精修后最终自由R因子为20.8%。对于磷酸盐结合形式，收集到分辨率为2.00 Å的数据集。空间群也是C222(1)，晶胞参数为a = 121.96、b = 137.61、c = 62.23 Å。该结构具有先前报道的FBPA结构典型的桶状三级结构，并且在活性位点呈现相同的席夫碱。四级结构为二聚体。这项工作为兔脑炎微孢子虫FBPA产物状态的底物结合构象提供了直接的实验结果。
