Prompers J J, Groenewegen A, Van Schaik R C, Pepermans H A, Hilbers C W
Nijmegen SON Research Center, Laboratory of Biophysical Chemistry, University of Nijmegen, The Netherlands.
Protein Sci. 1997 Nov;6(11):2375-84. doi: 10.1002/pro.5560061111.
Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.
利用异核核磁共振技术,对在大肠杆菌中作为214个残基的异源蛋白产生的茄病镰刀菌角质酶的主链和侧链1H、13C和15N共振进行了基本完整(96%)的序列特异性归属。在归属过程中注意到三个结构特征。(1)溶液中的二级结构大多与X射线衍射得到的结构一致,这表明两种结构在整体上是相似的。(2)Ala32的HN在3.97 ppm处有一个强烈的高场位移共振,表明与Trp69的吲哚环形成了酰胺-芳香氢键,该氢键稳定了平行β-折叠的N端侧。(3)核磁共振数据表明,与晶体结构中观察到的预先形成的氧阴离子洞不同,构成氧阴离子洞的残基在溶液中的游离酶中相当灵活。显然,角质酶像真正的脂肪酶一样,在结合底物时形成其氧阴离子洞。