Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.
Int J Biol Macromol. 2018 Apr 1;109:980-991. doi: 10.1016/j.ijbiomac.2017.11.087. Epub 2017 Nov 17.
The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α) incomplete toroidal fold and a layered β sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% β sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×10) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during β-elimination.
特征描述了 8 家族多糖裂解酶家族的软骨素 AC 裂解酶（PsPL8A）的结构。建模的 PsPL8A 结构显示，它包含 N 端（α/α）不完整的环形折叠和 C 端的层状 β 三明治结构。Ramchandran 图显示 98.5%的残基处于有利区域，1.2%的残基处于允许区域。CD 显示 PsPL8A 的二级结构为 27.31%的α螺旋、22.7%的β片层和 49.9%的无规卷曲。蛋白质融解研究表明，PsPL8A 在 60°C 时完全展开。SAXS 分析表明，PsPL8A 在溶液形式下完全折叠。从头计算得到的 PsPL8A 虚拟模型与模型结构很好地叠加，除了一些α螺旋和环区。结构叠加和对接分析表明，N153、W105、H203、Y208、Y212、R266 和 E349 参与催化。SDM 产生的突变体 N153A、H203A、Y212F、R266A 和 E349A 没有残留活性。与 C4S 多糖的 Y212F 和 H203A 的等温滴定量热分析表明，Y212F 具有中等结合（Ka=9.56±3.81×10），而 H203A 没有结合，表明 Y212 在底物结合中具有活性贡献。Y212 和 H203 或 R266 残基可能分别作为广义碱和广义酸。N153 和 E349 残基可能有助于电荷中和，并在β-消除过程中稳定烯醇阴离子中间体。
