High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop.
High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop.
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DOI：
10.4331/wjbc.v2.i5.90
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发表时间：
2011-05-26
期刊：
影响因子：
--
通讯作者：
Fan, Huizhou
中科院分区：
文献类型：
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作者：
Oey, Christopher B;Bao, Xiaofeng;Fan, Huizhou
目的：研究沙眼衣原体多肽脱甲酰基酶(PDF)的环区是否以及如何调控酶的功能。方法：采用分子动力学模拟方法，研究沙眼衣原体多肽脱甲酰基酶(CPDF)的结构模型，预测蛋白质骨架原子的单位残基温度因子。采用定点突变技术构建cPDF突变体。结果：在电子计算机分析中预测，与野生型cPDF相比，cPDF突变体中环区域的DGELV序列(残基68-72)的原子运动显著增加，与野生型cPDF相比，由于活性部位附近的两个氨基酸取代，cPDF突变体对PDF抑制剂具有抗药性。D68R和D68R/E70R cPDF突变体显著提高了催化效率。E70R突变体仅表现出轻微的效率下降。虽然68-72残基的缺失导致底物结合减少近3倍，但催化效率的提高弥补了这一不足。结论：DGELV环区的移动参与了酶在催化过程中限速构象的变化。然而，对于cPDF酶活性，该区域没有严格的序列要求。
AIM: To determine if and how a loop region in the peptide deformylase (PDF) of Chlamydia trachomatis regulates enzyme function.METHODS: Molecular dynamics simulation was used to study a structural model of the chlamydial PDF (cPDF) and predict the temperature factor per residue for the protein backbone atoms. Site-directed mutagenesis was performed to construct cPDF variants. Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate.RESULTS: In silico analysis predicted a significant increase in atomic motion in the DGELV sequence (residues 68-72) of a loop region in a cPDF mutant, which is resistant to PDF inhibitors due to two amino acid substitutions near the active site, as compared to wild-type cPDF. The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency. The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding, this deficiency was compensated for by increased catalytic efficiency.CONCLUSION: Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity.