Siderophores are critical virulence factors for pathogenic bacteria, and the structure of malleobactin, the siderophore of the human pathogenic Burkholderia mallei family (B. mallei and B. pseudomallei), had remained unresolved. Here we report the unusual structure and absolute configuration of malleobactin A and reveal the biogenetic origin of its unprecedented aliphatic nitro amino acid component. By comparing ornibactin (hydroxamate siderophore from Burkholderia cepacia complex) and malleobactin biosynthesis gene clusters, we found the latter lacks acyltransferase genes (orbK/L), suggesting structural differences. Using Burkholderia thailandensis (a less virulent model), we generated knock-out mutants (ΔpchE for pyochelin, ΔmbaA for malleobactin) and validated via CAS assays (Fe³⁺ binding) and LC-MS that both clusters are essential for siderophore activity. Structural elucidation of malleobactin A via 2D NMR and MS showed it contains 2-amino-5-nitropentanoic acid (ANPA), an aliphatic nitro amino acid never before reported in a natural product. Absolute configuration analysis (Marfey's method) revealed malleobactin A features N-formylated L-ANPA instead of the N-acylated ornithine found in ornibactins. Biochemical studies of the N-hydroxylase MbaC demonstrated it catalyzes ornithine N-hydroxylation, producing a hydroxylamine intermediate (malleobactin B) that spontaneously oxidizes to a nitroso derivative (malleobactin C), the nitro compound malleobactin A, and an azoxy-linked dimer (malleobactin D). The absence of N-acylation (due to missing orbK/L) leaves the hydroxylamine unprotected, leading to over-oxidation to the nitro group. Our work elucidates malleobactin's structure, identifies ANPA as a novel natural nitro amino acid, and reveals a biosynthetic route to aliphatic nitro groups: enzymatic N-hydroxylation followed by spontaneous oxidation. We also show that N-acylation in hydroxamate siderophores protects N-hydroxy groups from over-oxidation, a key functional role beyond iron coordination.