Soe Cho Z, Codd Rachel
School of Medical Sciences (Pharmacology) and Bosch Institute, The University of Sydney , Sydney, New South Wales 2006, Australia.
ACS Chem Biol. 2014 Apr 18;9(4):945-56. doi: 10.1021/cb400901j. Epub 2014 Feb 5.
To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; M + H(+), m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4-diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of l-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, (15)N2-1,4-diaminobutane ((15)N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of (15)N-labeled pbH2 ((15)N4-pbH2; M + H(+), m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; M + H(+), m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; M + H(+), m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 (Fe(pb); M, m/zcalc 426.1, m/zobs 426.2), (15)N4-pbH2 (Fe((15)N4-pb); M, m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 (Fe(E,E-pbe); M, m/zcalc 422.1, m/zobs 422.0), or E-pbxH2 (Fe(E-pbx); M, m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical conversions across the double bond(s) of the unsaturated macrocycles provides a new route to increased molecular diversity in this class of siderophore.
为获取生长所需的铁，腐败希瓦氏菌产生大环二氧肟酸腐铁菌素（pbH2；M + H(+)，计算质荷比m/z 373.2）作为其天然铁载体。pbH2的组装需要内源性1,4 - 二氨基丁烷（DB），它由鸟氨酸脱羧酶（ODC）催化L - 鸟氨酸脱羧产生。在本研究中，通过在腐败希瓦氏菌培养物的培养基中添加ODC抑制剂1,4 - 二氨基 - 2 - 丁酮（DBO）来降低内源性DB的水平。培养基中存在DBO以及替代的外源性非天然二胺底物，(15)N2 - 1,4 - 二氨基丁烷((15)N2 - DB)或1,4 - 二氨基 - 2(E) - 丁烯（E - DBE），分别导致了(15)N标记的pbH2（(15)N4 - pbH2；M + H(+)，计算质荷比m/z 377.2，实测质荷比m/z 377.2）或不饱和pbH2变体（在此命名为：E,E - 腐铁烯（E,E - pbeH2；M + H(+)，计算质荷比m/z 369.2，实测质荷比m/z 369.2））的生物合成。在后一种体系中，剩余的内源性DB导致单不饱和DB - E - DBE杂合物E - 腐铁烯（E - pbxH2；M + H(+)，计算质荷比m/z 371.2，实测质荷比m/z 371.2）的平行生物合成。这些是首次鉴定出的不饱和大环二氧肟酸铁载体。液相色谱 - 质谱测量显示Fe(III)与pbH2（Fe(pb)；M，计算质荷比m/z 426.1，实测质荷比m/z 426.2）、(15)N4 - pbH2（Fe((15)N4 - pb)；M，计算质荷比m/z 430.1，实测质荷比m/z 430.1）、E,E - pbeH2（Fe(E,E - pbe)；M，计算质荷比m/z 422.1，实测质荷比m/z 422.0）或E - pbxH2（Fe(E - pbx)；M，计算质荷比m/z 424.1，实测质荷比m/z 424.2）形成1:1的配合物。根据游离配体与负载Fe(III)的配合物之间保留时间的差异所定义的铁载体介导的Fe(III)溶解度增加的顺序为pbH2（ΔtR = 8.77分钟）＞E - pbxH2（ΔtR = 6.95分钟）＞E,E - pbeH2（ΔtR = 6.16分钟），这表明自然界选择饱和而非不饱和铁载体作为Fe(III)增溶剂的一个可能原因。对不饱和大环双键进行多种类型的非原位化学转化的潜力为增加这类铁载体的分子多样性提供了一条新途径。
