Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.
Crystal structure of tyrosine decarboxylase and identification of key residues involved in conformational swing and substrate binding.
复制标题
酪氨酸脱羧酶的晶体结构以及参与构象摆动和底物结合的关键残基的鉴定
DOI：
10.1038/srep27779
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发表时间：
2016-06-13
影响因子：
4.6
通讯作者：
Ni Y
中科院分区：
文献类型：
--
作者：
Zhu H;Xu G;Zhang K;Kong X;Han R;Zhou J;Ni Y
酪氨酸脱羧酶(TDC)是一种依赖于5-磷酸吡哆醛(PLP)的酶，主要负责合成重要的生物胺--酪胺。本研究测定了短链乳杆菌(LbTDC)apo和holo构型的晶体结构。LbTDC与唯一报道的TDC结构仅显示25%的序列同源性。对构象柔性中心和催化中心进行了定点突变，以探讨其潜在的催化机制。发现活性中心的H_241在PLP结合中起着重要作用，因为它在LbTDC的apo和holo结构中具有不同的构象。与PLP结合后，H241旋转到与PLP吡啶环相邻的位置。丙氨酸扫描突变揭示了决定底物特异性和催化活性的几个关键区域。在突变体中，S586A突变体表现出更高的催化效率和底物亲和力，这归因于空间位阻的减少和疏水性的增加，S586的饱和突变证实了这一点。我们的结果提供了关于残基的结构信息，这些残基对于TDC的蛋白质工程在提高酪胺绿色制造中的催化效率方面具有重要意义。
Tyrosine decarboxylase (TDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme and is mainly responsible for the synthesis of tyramine, an important biogenic amine. In this study, the crystal structures of the apo and holo forms ofLactobacillus brevisTDC (LbTDC) were determined. TheLbTDC displays only 25% sequence identity with the only reported TDC structure. Site-directed mutagenesis of the conformationally flexible sites and catalytic center was performed to investigate the potential catalytic mechanism. It was found that H241 in the active site plays an important role in PLP binding because it has different conformations in the apo and holo structures ofLbTDC. After binding to PLP, H241 rotated to the position adjacent to the PLP pyridine ring. Alanine scanning mutagenesis revealed several crucial regions that determine the substrate specificity and catalytic activity. Among the mutants, the S586A variant displayed increased catalytic efficiency and substrate affinity, which is attributed to decreased steric hindrance and increased hydrophobicity, as verified by the saturation mutagenesis at S586. Our results provide structural information about the residues important for the protein engineering of TDC to improve catalytic efficiency in the green manufacturing of tyramine.