Unsaturated Macrocyclic Dihydroxamic Acid Siderophores Produced by Shewanella putrefaciens Using Precursor-Directed Biosynthesis

To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH(2); [M + H+](+), m/z(calc) 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, N-15(2)-1,4-diaminobutane (N-15(2)-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of N-15-labeled pbH(2) (N-15(4)-pbH(2); [M + H+](+), m/z(calc) 377.2, m/z(obs) 377.2) or the unsaturated pbH(2) variant, named here: E,E-putrebactene (E,E-pbeH2; [M + m/z(calc) 369.2, m/z(obs) 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH(2); [M + H+](+) m/z(calc) 371.2, m/z(obs) 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC-MS measurements showed 1:1 complexes formed between Fe(III) and pbH(2) ([Fe(pb)](+); [M](+), m/z(calc) 426.1, m/z(obs) 426.2), N-15(4-)-pbH(2) ([Fe(N-15(4)-pb)](+); [M](+), m/z(calc) 430.1, m/z(obs) 430.1), E,E-pbeH(2) ([Fe(E,E-pbe)](+); [M](+), m/z(calc) 422.1, m/z(obs) 422.0), or E-pbxH(2) ([Fe(E-pbx)](+); [M](+), m/z(calc) 424.1, m/z(obs) 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 pbH(2) (Delta t(R) = 8.77 min) > E-pbxH(2) (Delta t(R) = 6.95 min) > E,E-pbeH(2) (Delta t(R) = 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.