Biosynthesis of desferrioxamine siderophores initiated by decarboxylases: A functional investigation of two lysine/ornithine-decarboxylases from Gordonia rubripertincta CWB2 and Pimelobacter simplex 3E
Biosynthesis of desferrioxamine siderophores initiated by decarboxylases: A functional investigation of two lysine/ornithine-decarboxylases from Gordonia rubripertincta CWB2 and Pimelobacter simplex 3E
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DOI：
10.1016/j.abb.2020.108429
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发表时间：
2020-08-15
影响因子：
3.9
通讯作者：
Tischler, Dirk
中科院分区：
文献类型：
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作者：
Hofmann, Marika;del Campo, Julia S. Martin;Tischler, Dirk
赖氨酸是去铁胺铁载体生物合成的前体。该途径通常由赖氨酸脱羧酶起始。然而，很少有人知道这些酶从放线菌，代表了不同类别的去铁胺生产商。在这项研究中，我们专注于基因grdesA形式Gordonia rubipertincta CWB 2和psdesA从Pimelelopsis simplex VkMAC-2033 D，编码脱羧酶，推测参与合成去铁胺铁载体。相应的蛋白质GrDesA和PsDesA，在大肠杆菌中异源产生并纯化。分离PsDesA与辅因子吡哆醛5-磷酸结合，并以其载脂蛋白形式纯化GrDesA。PsDesA对赖氨酸表现出中等的底物偏好性（K-m = 0.17 mM，k（cat）= 0.26 s（-1））与鸟氨酸相比（K-m = 0.13 mM，k（cat）= 0.14 s（-1）），而GrDesA对赖氨酸表现出特异性（K-m = 0.13 mM，k（cat）= 1.2 s（-1））与鸟氨酸（K-m = 2.9 mM，k（cat）= 0.18 s（-1））相比。PsDesA的最大脱羧酶活性在35 ℃、pH7.5时达到，尽管PsDesA在40 ℃时仍保持稳定，但在50 ℃时其相对活性显著下降。GrDesA的最适温度（40 ℃）和热稳定性也是如此，但它在pH范围8.0-8.5时表现出最大活性，在此范围外急剧下降。这两种脱羧酶的表达和特性为研究G. rubripertincta和P. simplex，并支持相关途径的功能注释。
Lysine is a precursor for desferrioxamine siderophore biosynthesis. The pathway is often initiated by lysine decarboxylases. However, little is known about those enzymes from Actinobacteria which represents a diverse class of desferrioxamine producers. In this study we focused on the genes grdesA form Gordonia rubripertincta CWB2 and psdesA from Pimelobacter simplex VkMAC-2033D that encode decarboxylases presumed to be involved in the synthesis of desferrioxamine siderophores. The corresponding proteins GrDesA and PsDesA, were heterologously produced in Escherichia coli and purified. PsDesA was isolated bound to the cofactor pyridoxal 5-phosphate and GrDesA was purified in its apo form. PsDesA showed a moderate substrate preference for lysine (K-m = 0.17 mM, k(cat) = 0.26 s(-1)) compared to ornithine (K-m = 0.13 mM, k(cat) = 0.14 s(-1)), while GrDesA exhibited specificity for lysine (K-m = 0.13 mM, k(cat) = 1.2 s(-1)) compared to ornithine (K-m = 2.9 mM, k(cat) = 0.18 s(-1)). The maximum decarboxylase activity of PsDesA was achieved at pH 7.5 at 35 degrees C, although PsDesA was stable up to 40 degrees, its relative activity decreased significantly at 50 degrees C. The temperature optimum (40 degrees C) and thermostability of GrDesA were likewise, but it exhibited maximum activity at pH range 8.0-8.5, and sharply decreased outside of this range. The expression and characterization of these two decarboxylases provides insight into the biosynthetic pathway of desferrioxamines from G. rubripertincta and P. simplex and supports the functional annotation of related pathways.