Structural Modelling, Substrate Binding and Stability Studies of Endopectate Lyase (PL1B) of Family 1 Polysaccharide Lyase from Clostridium thermocellum.
Structural Modelling, Substrate Binding and Stability Studies of Endopectate Lyase (PL1B) of Family 1 Polysaccharide Lyase from Clostridium thermocellum.
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DOI：
10.2174/0929866522666150506141552
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发表时间：
2015-05
影响因子：
1.6
通讯作者：
S. Chakraborty;Kedar Sharma;J. Mukherjee;M. N. Gupta;A. Goyal
中科院分区：
文献类型：
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作者：
S. Chakraborty;Kedar Sharma;J. Mukherjee;M. N. Gupta;A. Goyal
对热纤梭菌（Clostridium thermocellum）第1家族多糖裂解酶的内切果胶酸裂解酶（PL 1B）进行了结构表征，并测定了其在离液剂作用下的稳定性。从属于果胶酸裂解酶超家族3的蛋白质序列ABN53381.1中鉴定出推定的结构域PL 1B。PL 1B与其他已知果胶酸裂解酶的多重序列比对揭示了保守和半保守的残基。PsiPred预测的PL 1B二级结构由2个α-螺旋（2.06%）、26个β-链（40.54%）和29个无规卷曲（57.4%）组成。模拟的蛋白质代表右手平行β-螺旋结构，其中三个平行β-折叠通过环连接以形成β-螺旋核心。利用Ramachandran图对能量最小化结构进行质量评价，结果显示82.8%的残留物在有利区域。将PL 1B结构与芽孢杆菌Bsp 165-PelA的结构进行叠加，结果表明，PL 1B结构中的氨基酸残基与β-折叠形成底物结合裂缝。分子动力学模拟的PL 1B结构推断，它是相当稳定和紧凑的。对接研究确定Asp 151、Arg 209、Asn 234、Arg 236、Tyr 271和Ser 272为参与催化的PL 1B的关键残基。其中Arg 209负责β-消除过程中的质子提取。对PL 1B的蛋白质熔融研究表明，在0.6 mM Ca（2+）离子存在下，峰从74 °C移动至86°C，这表明它们为结构提供了稳定性。通过荧光研究的GuHCl或尿素对PL 1B的解折叠表明，蛋白质结构是稳定的，并且在其较高浓度下分解。
An endo-pectate lyase (PL1B) of family 1 polysaccharide lyase from Clostridium thermocellum was structurally characterized and its stability under chaotropic agent was determined. The putative domain PL1B was identified from the protein sequence ABN53381.1 belonging to superfamily 3 of pectate lyase. Multiple sequence alignment of PL1B with other known pectate lyases revealed the conserved and semi-conserved residues. The secondary structure of PL1B predicted by PsiPred and confirmed by Circular Dichroism showed the presence of 2 α-helices (2.06%), 26 β-strands (40.54%) and 29 random coils (57.4%). The modelled protein represented right handed parallel β-helix structure, where three parallel β-sheets linked by loops coils around to form the β-helix core. Quality assessment of energy minimized structure by Ramachandran plot displayed 82.8% residues in favoured region. Superposition of PL1B structure with Bsp165-PelA from Bacillus sp. revealed the substrate binding cleft formed by the amino acid residues from the loops and β-sheet. Molecular dynamic simulation of modelled PL1B structure inferred that it is quite stable and compact. Docking studies identified Asp151, Arg209, Asn234, Arg236, Tyr271 and Ser272 as the key residues of PL1B involved during catalysis. Among them Arg209 is responsible for proton abstraction during β-elimination. Protein melting studies on PL1B showed that there was 12°C shift of peak from 74 to 86°C in presence of 0.6 mM Ca(2+) ions, showing that they provide stability to the structure. The unfolding of PL1B by GuHCl or Urea by fluorescence study showed that the protein structure is stable and disintegrates at their higher concentrations.